Promoter

ABSTRACT

A promoter is described. The promoter comprises a nucleotide sequence corresponding to that shown as SEQ ID No. 1 or a variant, homologue or derivative thereof.

FIELD OF THE PRESENT INVENTION

The present invention relates to a promoter, including a construct andan expression vector comprising the same and a transformed cellcomprising the same. In addition the present invention relates to aplant cell, as well as a plant, comprising the same.

BACKGROUND OF THE PRESENT INVENTION

Expression of plant genes is controlled in a complex pattern during thelife cycle of a plant. Several processes are involved in this regulationof gene expression. The main steps are; the initiation of transcription;the termination of transcription; the processing of transcripts; thetransport of mRNA to the ribosomes; and the translation.

Three examples of plant genes that are expressed are found in Huang etal (1996 Biosci Biotech Biochem 60 (2) 233–239) who report on three ricesucrose synthase isogenes, which are called RSus1, RSus2 and RSus3. Theauthors also report on their differential regulation of theirexpression. The authors state that the gene organisation patterns ofRSus2 and RSus3 were the same.

One of the major processes controlling gene expression is initiation oftranscription. The transcription is initiated by binding of RNApolymerase together with several transcription factors to the promoterregion. Specific regulatory DNA sequences (cis-elements) in the promoterserve as binding sites for these transcription factors (trans-actingfactors).

The cis-elements found in plant gene promoters can be divided in twocategories. The first category comprises those cis-elements which areinvolved in initiation of transcription. The TATA box and the CAAT boxare examples of proximal cis-elements involved in initiation oftranscription. The CAAT box defines the binding site for the RNApolymerase, and the TATA box directs the RNA polymerase to the correcttranscription start site. Presence of multiple CAAT boxes normallyindicate a constitutive promoter. The TATA box and CAAT box areconserved among prokaryotes and eukaryotes, but are not essential forthe function of some plant gene promoters. The second category iscomposed of cis-elements which are involved in temporal and spatialregulation of gene expression. Genes encoding seed storage proteins(Glutamins, Legumins, Prolamins etc.) are examples of genes which aretemporally and spatially regulated, and are thereby expressed in atissue-specific and developmental manner. Examples of endosperm-specificcis-elements are the AACA motif and the endosperm box. Thesecis-elements, that contribute to tissue-specific and developmentalexpression of endosperm storage protein genes, are conserved among awide range of seed storage protein genes.

The manner in which complex patterns of transcription factors act onspecific cis-elements determines whether genes are more or lessconstitutively expressed, or are expressed at specific times duringdevelopment. Furthermore, these interactions determine whetherexpression occurs in a specific tissue e.g. the endosperm, is displayedby several tissues e.g. those of the seed, or is common to all parts ofthe plant. Multiple trans-acting factors can recognise variants of thecis-element consensus sequences and compete for binding, yieldingcomplex expression patterns. Each plant gene promoter has a set oftranscription factors and other trans-acting factors, which interactwith the promoter sequence and the RNA polymerase and thereby regulategene expression in an unique pattern.

It is known that it is desirable to direct expression of a nucleotidesequence of interest (“NOI”) in certain tissues of an organism—such as aplant. The NOI will typically encode a product of interest (“POI”). Forexample, it may be desirable to produce crop protein products with anoptimised amino acid composition and so increase the nutritive value ofthe crop. It may even be desirable to use the crop to express non-plantgenes such as genes for mammalian products. Examples of the latterproducts include interferons, insulin, blood factors and plasminogenactivators.

However, whilst it may be desirable to achieve expression of a NOI incertain tissues it is sometimes important (if not necessary) to ensurethat the NOI is not expressed in other tissues in such a manner thatdetrimental effects may occur. Moreover, it is important not to upsetthe normal metabolism of the organism to such an extent that detrimentaleffects occur. For example, a disturbance in the normal metabolism in aplant's leaf or shoot could lead to stunted growth of the plant.

An example of the use of plant promoters to cause expression of an NOIin plant tissue may be found in CA-A-2006454, which describes a DNAsequence of an expression cassette in which the potato tuber specificregulatory regions are localised. The expression cassette contains apatatin-gene with a patatin-gene promoter. The DNA sequence istransferred into a plant genome using Agrobacterium. According toCA-A-2006454, the DNA sequence enables heterologous products to beprepared in crops.

However, in plant transformation processes, it is generally the lowefficiency of both transformation and regeneration that seriously slowvector development, since they limit the number of genetic constructswhich can be tested. Investigations of the strength and tissuespecificity of different transcriptional promoters, which can greatlyinfluence the effect of the genetic manipulation, can be unmanageablylabour-intensive if performed in stable transformants. This has resultedin a tendency to use strong constitutive promoters which are nottissue-specific to direct transgenic expression in stable transformants,e.g. from viruses (cauliflower mosaic virus 35S promoter) andAgrobacterium (nopaline synthase promoter (NOS)). Not the leastproblematic element of this approach occurs when lowering of geneexpression by antisense transcription is attempted, because gratuitoussuppression of gene expression in all tissues can result indevelopmental retardation of non-target tissues, or other weakening ofthe transformant. Under such circumstances, only those transformants, inwhich the genetic construct poorly suppresses gene expression, would beexpected to survive selection. There is, therefore, a strong argumentfor the use of tissue-specific transcriptional promoters for directingantisense transcription, but, in many crop species, characterising suchpromoters in stable transformants is not feasible owing to lowefficiency of transformation and regeneration.

Despite the fact that there are already some promoters available in theart, there is still a need to have additional promoters, in particularpromoters that are efficient and/or selective in their ability to allowfor the expression of a NOI.

Thus, the present invention seeks to provide a promoter that is capableof causing the expression (transcription) of a NOI.

More in particular, the present invention seeks to provide a promoterthat is capable of directing the expression (transcription) of anucleotide sequence of interest in specific tissues, or in just aspecific tissue, of an organism, typically a plant.

SUMMARY ASPECTS OF THE PRESENT INVENTION

Aspects of the present invention are presented in the claims and in thefollowing commentary.

In brief, some aspects of the present invention relate to:

-   1. A novel promoter capable of selective expression.-   2. Novel promoter nucleotide sequences.-   3. Expression systems comprising said promoters.-   4. Methods of expression using said promoters.-   5. Transformed hosts/host cells comprising said promoters.

As used with reference to the present invention, the terms “expression”,“expresses”, “expressed” and “expressable” are synonymous with therespective terms “transcription”, “transcribes”, “transcribed” and“transcribable”. Hence, if the NOI is a coding sequence, then theproduct of its expression may also be called the transcription productand visa versa. Likewise, if the NOI is an anti-sense nucleotidesequence then the product of its transcription may also be called theexpression product and visa versa.

In the following commentary references to “nucleotide sequence of thepresent invention” include references to the “promoter of the presentinvention” and vice versa. Also, the term “nucleotide sequence of thepresent invention” is synonymous with the phrase “polynucleotidesequence of the present invention”.

Other aspects concerning the promoter and/or the nucleotide sequence ofthe present invention include: a construct comprising the sequences ofthe present invention; a vector comprising the sequences of the presentinvention; a plasmid comprising the sequences of present invention; atransformed cell comprising the sequences of the present invention; atransformed tissue comprising the sequences of the present invention; atransformed organ comprising the sequences of the present invention; atransformed host comprising the sequences of the present invention; atransformed organism comprising the sequences of the present invention.The present invention also encompasses methods of expressing NOIs usingthe same, such as expression in a host plant cell; including methods fortransferring same.

For convenience, the promoter of the present invention is sometimesreferred to as the RSus3 promoter (or even the Rsus3 promoter or eventhe RSus3 promoter). However, it is important to note that the promoterof the present invention is not disclosed in the teachings of Huang etal (ibid). Moreover, and contrary to the authors statement that “thegene organisation patterns of RSus2 and RSus3 were the same” we foundthat the promoter of RSus3 was quite different to that of RSus2.

In this respect, we found a very low degree of homology (i.e. identity)between the RSus3 promoter and the RSus2 promoter and also the RSus3 andRSus1 promoter—as evidenced by the following Table.

Homology scores of RSus1, RSus2, RSus3 Alignment % Homology¹ % Homology²RSus3 and RSus1 7.7% 5.4% RSus3 and RSus2 4.6% 5.4% RSus1 and RSus2 4.6%8.8% ¹Homology scores based on multiple alignment (all of the promoterregion upstream of the translational start codon including the intron:RSus1 (2663 bp), RSus2 (2900 bp), RSus3 (2667 bp)). ²Homology scoresbased on multiple alignment (RSus1, RSus2, RSus3 following excision ofintron 1).

The homology scores presented in the above table show the low degree ofsimilarity between the promoter regions of RSus1, RSus2 and RSus3.

The percentage homologies were calculated using the multiple alignmentfeature in DNASIS™ (Hitachi Software), based on an algorithm, analogousto CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237–244).

In addition, and as a result of a sequence analysis of the sequences ofthe RSus3 promoter and the RSus1 promoter and the RSus2 promoter, wefound that apart from the conserved TATA box and intron splice sites,they have no motifs in common.

Thus, contrary to the prior art teachings, we found that the geneorganisation patterns of RSus2 and RSus3 are not the same.

For ease of reference, aspects of the present invention are nowdiscussed under appropriate section headings. However, the teachingsunder each section are not necessarily limited to each particularsection.

DETAILED ASPECTS OF THE PRESENT INVENTION

According to a first aspect of the present invention there is provided apromoter having the nucleotide sequence presented as SEQ ID No. 1, or avariant, homologue, fragment or derivative thereof.

Alternatively expressed, the present invention provides a nucleotidesequence selected from:

-   (a) the nucleotide sequence presented as SEQ ID No. 1;-   (b) a nucleotide sequence that is a variant, homologue, derivative    or fragment of the nucleotide sequence presented as SEQ ID No. 1;-   (c) a nucleotide sequence that is the complement of the nucleotide    sequence set out in SEQ ID No. 1;-   (d) a nucleotide sequence that is the complement of a variant,    homologue, derivative or fragment of the nucleotide sequence    presented as SEQ ID No. 1;-   (e) a nucleotide sequence that is capable of hybridising to the    nucleotide sequence set out in SEQ ID No. 1;-   (f) a nucleotide sequence that is capable of hybridising to a    variant, homologue, derivative or fragment of the nucleotide    sequence presented as SEQ ID No. 1;-   (g) a nucleotide sequence that is the complement of a nucleotide    sequence that is capable of hybridising to the nucleotide sequence    set out in SEQ ID No. 1;-   (h) a nucleotide sequence that is the complement of a nucleotide    sequence that is capable of hybridising to a variant, homologue,    derivative or fragment of the nucleotide sequence presented as SEQ    ID No. 1;-   (i) a nucleotide sequence that is capable of hybridising to the    complement of the nucleotide sequence set out in SEQ ID No. 1;-   (j) a nucleotide sequence that is capable of hybridising to the    complement of a variant, homologue, derivative or fragment of the    nucleotide sequence presented as SEQ ID No. 1;-   (k) a nucleotide sequence comprising any one of (a), (b), (c), (d),    (e), (f), (g), (h), (i), and/or (j).

In a preferred aspect, the promoter is obtainable from (though it doesnot have to be actually obtained from) a plant of the genus Oryza,preferably from Oryza sativa.

Another aspect of the present invention includes an isolated nucleotidesequence according to the present invention.

Additional aspects of the present invention include uses of the promoterfor expressing NOIs in vitro (e.g. in culture media such as a broth)and/or in vivo (e.g. in a transformed organism).

In a highly preferred aspect the present invention provides a method forexpressing an NOI in endosperm (preferably selectively expressing inendosperm), the method comprising expressing the NOI when it is operablylinked to the promoter of the present invention.

The terms “selective” and “selectively” as used herein with respect tothe present invention are synonymous respectively with the terms“specific” and “specifically”. In this respect, selective or specificexpression by the promoter of the present invention in the endospermlayer means that higher levels of expression occur in the endospermrelative to other tissue types or cells.

In some instances, the NOI is highly selectively expressed in theendosperm. In this respect, highly selective or specific expression bythe promoter of the present invention in the endosperm layer means thatexpression occurs predominantly in the endosperm relative to othertissue types or cells and in some cases almost exclusively in theendosperm layer.

Preferably the transformed host/host cells is/are plant/plant cells.

The plant can be any suitable monocot plant (such as maize) or anysuitable dicot plant (such as soya or guar).

Preferably the plant is an endospermous cereal or a legume.

In a preferred aspect, the plant is a member of the grass family—such asany one of wheat, maize, barley, oats, rye, or rice.

In an alternative preferred aspect, the plant is a legume—such as anyone of guar or locus bean.

Hence, preferably, the transformed host is a transformed member of thetaxonomic groups Gramineae (which may be known as Poaceae) orLeguminosae (which may be known as Fabaceae) or is a cell or tissuethereof.

Hence, preferably, the transformed plant is a transformed grass or atransformed legume.

Also, preferably, the transformed plant cell is a transformed grass cellor a transformed legume cell.

The present invention is advantageous for a number of reasons.

By way of example, the present invention is advantageous becausedesirable levels of the expression product of a NOI can be obtained.Here, the NOI expression product may be, for example, a desired compoundof benefit to humans or animals, e.g. a desirable foodstuff or an enzymehaving a beneficial effect, such as a foodstuff processing effect oreven a pharmaceutical effect. Furthermore, that product may be easilyretrievable.

Alternatively, the NOI expression product may affect metabolism withinthe host. In some cases, the NOI expression product may be a componentthat is essential for metabolism within the host. In these instances, itmay not be important for the POI to be retrieved from the host. In somecases, it may be important that the POI is not retrieved from the host.

The present invention is also advantageous because it allows transformedplants or plant cells to express desirable levels of the product ofexpression of a NOI.

The present invention is also advantageous because it allows transformedplants or plant cells to express in selective tissues or cell typesdesirable levels of the expression product of a NOI.

The promoter of the present invention is further advantageous as it canprovide good expression levels of a NOI under conditions of transientexpression in plant cells.

In a preferred aspect, the promoter is linked to the sequence presentedas SEQ ID No. 2, or a variant, homologue, derivative or fragmentthereof. The term “linked” includes direct or indirect (such as with theprovision of suitable spacer sequence(s)) linkages.

Preferably the sequence presented as SEQ ID No. 2, or a variant,homologue, derivative or fragment thereof is located in between thepromoter of the present invention and the NOI.

In this respect, we have found that in some transformed hosts, such astransformed plants (such as transformed guar), expression levels of theNOI can be elevated. This is important if it is desirable to have suchan elevated expression.

The promoter may be used in conjunction with one or more otherexpression elements—which may be alternatively called “functionalelements”. These additional expression elements may be linked to thepromoter of the present invention. The term “linked” includes direct orindirect (such as with the provision of suitable spacer sequence(s))linkages.

The additional expression element may enhance expression or inhibitundesirable expression.

The additional expression element may even be a promoter, wherein thatpromoter may be the same as or different to the promoter of the presentinvention, or even a part thereof.

The present invention also encompasses repeating units of promoters—suchas tandem repeats that comprise at least two promoter elements—one ofwhich will be the promoter of the present invention—such as threepromoter elements.

Preferably the additional expression elements are located intermediatethe promoter of the present invention and the NOI. Should it bedesirable, the additional expression elements and the promoter of thepresent invention may be separated by suitable restriction sites.

In this respect, we have found that in some transformed hosts, such astransformed plants (such as transformed guar), expression levels of theNOI can be elevated. This is important if it is desirable to have suchan elevated expression.

In one aspect, the NOI is an antisense nucleotide sequence.

In this respect, preferably the NOI is a sequence that is antisense toall or part of the gene encoding an epimerase, in particular a UDPgalactose epimerase, more in particular a UDP galactose-4-epimerase (EC5.1.3.3).

If the NOI is a sequence that is antisense to all or part of the geneencoding a UDP galactose epimerase—more in particular a UDPgalactose-4-epimerase (EC 5.1.3.3)—then expression of that antisensesequence by the promoter of the present invention could affect thegalactose units (such as causing a decrease thereof) on a galactomannanor other extracellular polysaccharides.

In an alternative aspect, the NOI is a sense nucleotide sequence.

Here, the NOI may be a sequence that constitutes all or part of the geneencoding a UDP galactose epimerase, more in particular a UDPgalactose-4-epimerase (EC 5.1.3.3).

Promoter

Thus, the present invention relates to a novel regulatory sequence,namely a promoter—which we have called the RSus3 promoter.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

The promoter may be the same as the naturally occurring form—for thisaspect, preferably the promoter is not present in its naturalenvironment. In addition, or in the alternative, the promoter is in anisolated and/or in a purified form. The promoter of the presentinvention can be a variant, homologue, fragment or derivative of thenaturally occurring promoter. The promoter can be obtainable from orproduced by any suitable source, whether natural or not, or it may besynthetic, semi-synthetic or recombinant.

A nucleotide sequence comprising the promoter of the present inventionand other associated nucleotide sequences is schematically presented inFIG. 1 (which is not to scale).

In this respect, the promoter sequence SEQ ID No. 1 is shown as Box A.As can be seen, Box A comprises two units—which have been showndiagramatically as Box B and Box C. Box B corresponds to SEQ ID No. 6and Box C corresponds to SEQ ID No. 4. SEQ ID No. 6 is the sequence upto the TATA box. SEQ ID No. 4 is the first part of an exon sequence.

In accordance with the present invention we have found that expressionmay still be achieved in some host cells with the promoter of thepresent invention in the absence of all or part of SEQ ID No. 4. In thisrespect, the promoter of the present invention would be represented asSEQ ID No. 6 and the comments concerning SEQ ID No. 1 would equallyapply to SEQ ID No. 6.

FIG. 1 also shows Box D—which corresponds to SEQ ID No. 2. SEQ ID No. 2is an intron sequence. In accordance with the present invention we havefound that surprisingly expression can still be achieved with thepromoter of the present invention in the absence of all or part of SEQID No. 2. Thus, in one embodiment of the present invention the promoterof the present invention is not used in conjunction with all or part ofSEQ ID No. 2.

However, in some instances we have surprisingly found that SEQ ID No. 2can elevate expression levels of an NOI and/or increase the selectivityof the expression. Hence, in an alternative preferred embodiment of thepresent invention the promoter of the present invention is used inconjunction with all or part of SEQ ID No. 2.

FIG. 1 also shows Box E—which corresponds to SEQ ID No. 5. SEQ ID No. 5is the second part of the exon sequence associated with SEQ ID No. C.For some applications, the promoter sequence of the present invention iscontained within a nucleotide sequence wherein SEQ ID No. 4 is fused toSEQ ID No. 5.

Thus, schematically, and by way of example, the promoter of the presentinvention may be represented as any one or more of: Box A; Box B with orwithout Box C; Box A with Box D; Box B—with or without Box C—with Box D;Box A with Box E; Box B—with or without Box C—with Box E; Box A with BoxD and Box E; Box B—with or without Box C—with Box D and Box E.

It is to understood that: Box A represents SEQ ID No. 1 or a variant,homologue, fragment or derivative thereof, preferably, Box A representsSEQ ID No. 1; Box B represents SEQ ID No. 6 or a variant, homologue,fragment or derivative thereof, preferably, Box B represents SEQ ID No.6; Box C represents SEQ ID No. 4 or a variant, homologue, fragment orderivative thereof, preferably, Box C represents SEQ ID No. 4; Box Drepresents SEQ ID No. 2 or a variant, homologue, fragment or derivativethereof, preferably, Box D represents SEQ ID No. 2; Box E represents SEQID No. 5 or a variant, homologue, fragment or derivative thereof,preferably, Box E represents SEQ ID No. 5.

In our analysis of the promoter of the present invention we haveidentified a number of interesting expression elements/functionalelements which resemble consensus sequences or parts thereof.

These identified sequences are presented in Table 1 below.

TABLE 1 Functional elements in Reference the RSus3 (Con- promoterPosition RSus3 Consensus sensus region Positon (-intron) sequence¹sequence sequence) Transla- +1 +1 CAATGG CAATGG [Joshi tion start (SEQID No (SEQ ID No 1987] site 7 8 Intron 1 −27 TCCAG|GC TGCAG|GT [Simpson(Acceptor (SEQ ID No (SEQ ID No & splice site) 9) 10) Filipowicz 1996],consensus for monocots Intron 1 −892 AG|GTAGA AG|GTAAGT (Donor G (SEQ ID(SEQ ID No splice site) No 11) 12) TATA −986 −121 TATAAAT TATATATA[Joshi box A (SEQ ID (SEQ ID No 1987] No 13) 14) CAAT −999 −134 GCACATTTGGNCAATC box T (SEQ ID T (SEQ ID No No 15) 16) GCN4 box −1032 −167GTGAGGC (G/A)TGA(G/ [Muller & AG (SEQ ID C)TCA(T/G) Knudsen No 17) (SEQID No 1993], cis- 18) element involved in endo- sperm specificity Endo-−1072 −207 AGTAAAG TG(T/C/A)A [Marzabal sperm (SEQ ID No AA(G/A) et al.boxes 19) (SEQ ID No 1998], cis- 20) element involved in endo- spermspecificity −1130 −265 TGCAAAC (SEQ ID No 21) −1349 −484 TGTCAAA (SEQ IDNo 22) Legumin −1539 −674 CGTGCAT CATGCATG [Baumlein boxes (RY G (SEQ ID(SEQ ID No et al. repeats) No 23) 24) 1992] cis- element involved inseed specificity −1586 −721 CATGTATG (SEQ ID No 25) −1624 −759 CATGCAT A(SEQ ID No 26) −1707 −842 CATGCAT GCATG (SEQ ID No 27) −2115 −1250CATGCCTG (SEQ ID No 28) −2480 −1615 CAGGCAT GCATC (SEQ ID No 29)

We currently believe that at least one or more, such as all, of thoseidentified sequences that are the same as or similar to the consensussequences should be present in the promoter sequence of the presentinvention.

As indicated above, the promoter can additionally include or be usedwith features to ensure or to increase expression in a suitable host.For example, the features can be conserved regions such as a Pribnow Boxor a TATA box.

The promoter may even contain or be used with other sequences to affect(such as to maintain, enhance, decrease) the levels of expression of thenucleotide sequence of the present invention. For example, suitableother sequences may include the Sh1-intron or an ADH intron. Othersequences may include inducible elements—such as temperature, chemical,light or stress inducible elements. Also, suitable elements to enhancetranscription or translation may be present. An example of the latterelement is the TMV 5′ signal sequence (see Sleat Gene 217 [1987]217–225; and Dawson Plant Mol. Biol. 23 [1993] 97).

The promoter of the present invention may be used in combination withone or more other expression elements or functional elements orregulatory elements.

The terms “expression elements”, “functional elements” and “regulatoryelements” include enhancers, expression regulation signals, secretionleader sequences, promoter consensus sequences, terminator sequences,and may even include other promoters.

The present invention also encompasses hybrid promoters that comprise atleast a part of the promoter of the present invention and at least apart of another promoter.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct.

As indicated above, the present invention also encompasses tandempromoters wherein at least one of the promoters is the promoter of thepresent invention. If the other promoter is a promoter of the presentinvention or part thereof then that combination may be called a tandemrepeat.

The other promoter may even be another promoter or part thereof. By wayof example, the other promoter may be selected for its efficiency indirecting the expression of the NOI in the desired expression host.

In one embodiment, a constitutive promoter may be selected to direct theexpression of the NOI. Such an expression construct may provideadditional advantages since it may circumvent the need to culture theexpression hosts on a medium containing an inducing substrate. Examplesof strong constitutive and/or inducible promoters which are preferredfor use in, for example, fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xInA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi),alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG—fromthe glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters. Examples of strong yeast promoters arethose obtainable from the genes for alcohol dehydrogenase,3-phosphoglycerate kinase and triosephosphate isomerase. Examples ofstrong bacterial promoters are the α-amylase and SP02 promoters as wellas promoters from extracellular protease genes. Examples of strong plantpromoters are the CaMV promoter and the SV40 35S promoter and the NOSpromoter.

For some applications, preferably the promoter is stably incorporatedwithin the transformed organism's genome.

The term “transformed” is synonymous with the term “transgenic”.

In a preferred aspect, the promoter is linked to the sequence presentedas SEQ ID No. 2, or a variant, homologue, derivative or fragmentthereof.

Naturally Occurring

As used herein “naturally occurring” refers to the promoter sequencefound in nature—i.e. the wild type promoter.

Isolated/Purified

As used herein, the terms “isolated” and “purified” refer to nucleicacid sequences, that are removed from their natural environment andisolated or separated from at least one other component with which theyare naturally associated.

Biologically Active

As used herein “biologically active” refers to a promoter according tothe present invention—such as a recombinant promoter—having a similarstructural function (but not necessarily to the same degree), and/orsimilar regulatory function (but not necessarily to the same degree),and/or similar biochemical function (but not necessarily to the samedegree) of the naturally occurring promoter. Specifically, a promoter ofthe present invention has the ability to express a NOI in endosperm,preferably selectively in endosperm.

Deletion

As used herein, a “deletion” is defined as a change in the nucleotidesequence in which one or more nucleotides are absent.

Insertion/Addition

As used herein, an “insertion” or “addition” is a change in thenucleotide sequence which has resulted in the addition of one or morenucleotides as compared to the naturally occurring promoter.

Substitution

As used herein, “substitution” results from the replacement of one ormore nucleotides or by one or more different nucleotides.

Variant/Homologue

The terms “variant” or “homologue” with respect to the nucleotidesequence of the present invention are synonymous with allelic variationsof the sequences.

In particular, the term “homology” as used herein may be equated withthe term “identity”. Here, sequence homology with respect to thenucleotide sequence of the present invention can be determined by asimple “eyeball” comparison (i.e. a strict comparison) of any one ormore of the sequences with another sequence to see if that othersequence has at least 75% identity to the sequence(s). Relative sequencehomology (i.e. sequence identity) can also be determined by commerciallyavailable computer programs that can calculate % homology between two ormore sequences. A typical example of such a computer program is CLUSTAL.

Sequence homology (or identity) may even be determined using anysuitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andare advantageously set to the defined default parameters.

Advantageously, “substantial homology” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (seehttp://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.The BLAST programs were tailored for sequence similarity searching, forexample to identify homologues to a query sequence. The programs are notgenerally useful for motif-style searching. For a discussion of basicissues in similarity searching of sequence databases, see Altschul et al(1994) Nature Genetics 6:119–129.

The five BLAST programs available at http://www.ncbi.nim.nih.gov performthe following tasks:

-   -   blastp compares an amino acid query sequence against a protein        sequence database;    -   blastn compares a nucleotide query sequence against a nucleotide        sequence database;    -   blastx compares the six-frame conceptual translation products of        a nucleotide query sequence (both strands) against a protein        sequence database;    -   tblastn compares a protein query sequence against a nucleotide        sequence database dynamically translated in all six reading        frames (both strands).    -   tblastx compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.    -   BLAST uses the following search parameters:    -   HISTOGRAM Display a histogram of scores for each search; default        is yes. (See parameter H in the BLAST Manual).    -   DESCRIPTIONS Restricts the number of short descriptions of        matching sequences reported to the number specified; default        limit is 100 descriptions. (See parameter V in the manual page).        See also EXPECT and CUTOFF.    -   ALIGNMENTS Restricts database sequences to the number specified        for which high-scoring segment pairs (HSPs) are reported; the        default limit is 50. If more database sequences than this happen        to satisfy the statistical significance threshold for reporting        (see EXPECT and CUTOFF below), only the matches ascribed the        greatest statistical significance are reported. (See parameter B        in the BLAST Manual).    -   EXPECT The statistical significance threshold for reporting        matches against database sequences; the default value is 10,        such that 10 matches are expected to be found merely by chance,        according to the stochastic model of Karlin and Altschul (1990).        If the statistical significance ascribed to a match is greater        than the EXPECT threshold, the match will not be reported. Lower        EXPECT thresholds are more stringent, leading to fewer chance        matches being reported. Fractional values are acceptable. (See        parameter E in the BLAST Manual).    -   CUTOFF Cutoff score for reporting high-scoring segment pairs.        The default value is calculated from the EXPECT value (see        above). HSPs are reported for a database sequence only if the        statistical significance ascribed to them is at least as high as        would be ascribed to a lone HSP having a score equal to the        CUTOFF value. Higher CUTOFF values are more stringent, leading        to fewer chance matches being reported. (See parameter S in the        BLAST Manual). Typically, significance thresholds can be more        intuitively managed using EXPECT.    -   MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX,        TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff &        Henikoff, 1992). The valid alternative choices include: PAM40,        PAM120, PAM250 and IDENTITY. No alternate scoring matrices are        available for BLASTN; specifying the MATRIX directive in BLASTN        requests returns an error response.    -   STRAND Restrict a TBLASTN search to just the top or bottom        strand of the database sequences; or restrict a BLASTN, BLASTX        or TBLASTX search to just reading frames on the top or bottom        strand of the query sequence.    -   FILTER Mask off segments of the query sequence that have low        compositional complexity, as determined by the SEG program of        Wootton & Federhen (1993) Computers and Chemistry 17:149–163, or        segments consisting of short-periodicity internal repeats, as        determined by the XNU program of Clayerie & States (1993)        Computers and Chemistry 17:191–201, or, for BLASTN, by the DUST        program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov).        Filtering can eliminate statistically significant but        biologically uninteresting reports from the blast output (e.g.,        hits against common acidic-, basic- or proline-rich regions),        leaving the more biologically interesting regions of the query        sequence available for specific matching against database        sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g., “XXXXXXXXX”).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Preferably, sequence comparisons are conducted using the simple BLASTsearch algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.

Other computer program methods to determine identify and similaritybetween the two sequences include but are not limited to the GCG programpackage (Devereux et al 1984 Nucleic Acids Research 12: 387 and FASTA(Atschul et al 1990 J Molec Biol 403–410).

Should Gap Penalties be used when determining sequence identity, thenpreferably the following parameters are used:

FOR BLAST GAP OPEN 0 GAP EXTENSION 0 FOR CLUSTAL DNA WORD SIZE 2 GAPPENALTY 10 GAP EXTENSION 0.1

Most preferably, sequence comparisons are conducted using DNASIS™.

As used herein, the terms “variant”, “homologue”, “fragment” and“derivative” embrace allelic variations of the sequences.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Hybridisation

The term “hybridisation” (sometimes written as “hybridization”) as usedherein shall include “the process by which a strand of nucleic acidjoins with a complementary strand through base pairing” (Coombs J (1994)Dictionary of Biotechnology, Stockton Press, New York N.Y.) as well asthe process of amplification as carried out in polymerase chain reaction(PCR) technologies as described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y.).

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Stringency of hybridisation refers to conditions under which polynucleicacids hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general, the stability of a hybrid is afunction of sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na+ at 65–68° C.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe).

High stringency typically occurs at about 5° C. to 10° C. below the Tmof the probe. High stringency conditions can be provided, for example,by hybridisation in an aqueous solution containing 6×SSC, 5× Denhardt's,1% SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/mldenatured salmon sperm DNA as non specific competitor. Followinghybridisation, high stringency washing may be done in several steps,with a final wash (about 30 min) at the hybridisation temperature in0.2–0.1×SSC, 0.1% SDS.

Moderate, or intermediate, stringency typically occurs at about 10° C.to 20° C. below the Tm of the probe.

Low stringency typically occurs at about 20° C. to 25° C. below the Tmof the probe.

As will be understood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences, while an intermediate (or low) stringency hybridization canbe used to identify or detect similar or related polynucleotidesequences.

Moderate stringency refers to conditions equivalent to hybridisation inthe above described solution but at about 60–62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove described solution at about 50–52° C. In that case, the final washis performed at the hybridisation temperature in 2× SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds.(1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).Optimal hybridisation conditions have to be determined empirically, asthe length and the GC content of the probe also play a role.

Nucleotide Sequence

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variants,homologues, fragments and derivatives thereof (such as portions thereof.The nucleotide sequence may be of genomic or synthetic or recombinantorigin which may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand.

Preferably, the term “nucleotide sequence” means DNA.

In a preferred embodiment, the nucleotide sequence per se of the presentinvention does not cover the native nucleotide sequence according to thepresent invention in its natural environment when it is linked to itsnaturally associated sequence(s) that is/are also in its/their naturalenvironment. For ease of reference, we shall call this preferredembodiment the “non-native nucleotide sequence”.

Typically, the nucleotide sequence of the present invention is preparedusing recombinant DNA techniques (i.e. recombinant DNA). However, in analternative embodiment of the invention, the nucleotide sequence couldbe synthesized, in whole or in part, using chemical methods well knownin the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser215–23, Horn T et al (1980) Nuc Acids Res Symp Ser 225–232).

The present invention also encompasses nucleotide sequences that arecomplementary to the sequences presented herein, or any derivative,fragment or derivative thereof. If the sequence is complementary to afragment thereof then that sequence can be used as a probe to identifysimilar sequences in other organisms etc.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences presented herein, or anyderivative, fragment or derivative thereof.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences that are complementary to thesequences presented herein, or any derivative, fragment or derivativethereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hydridising understringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015Na₃ citrate pH 7.0)) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC).

Advantageously, the invention provides nucleic acid sequences which arecapable of hybridising, under stringent conditions, to a fragment ofSEQ. ID. No. 1. Preferably, the fragment is between 15 and 50 bases inlength. Advantageously, it is about 25 bases in length.

By knowledge of the nucleic acid sequences set out herein it is possibleto devise partial and full-length nucleic acid sequences such as cDNAand/or genomic clones. Nucleic acid sequences obtained by PCR—such asfragments of the full length sequence, preferably fragments havingunique sequences—may then be used to obtain same or similar sequencesusing hybridization library screening techniques. The fragments may befrom 10 to 100 nucleotides long. Preferably, the fragments may be from15 to 90 nucleotides long. More preferably, the fragments may be from 20to 80 nucleotides long.

By way of example, a PCR clone may be labelled with radioactive atomsand used to screen a genomic library from other species, preferablyother plant species. Hybridization conditions will typically beconditions of medium to high stringency (for example 0.03M sodiumchloride and 0.03M sodium citrate at from about 50° C. to about 60° C.).

Degenerate nucleic acid probes encoding all or part of the amino acidsequence may also be used to probe cDNA and/or genomic libraries fromother species, preferably other plant species or fungal species.However, it is preferred to carry out PCR techniques initially to obtaina single sequence for use in further screening procedures.

Polynucleotide sequences of the present invention obtained using thetechniques described above may be used to obtain further homologoussequences and variants using the techniques described above.

Thus, polynucleotides of the present invention may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the present invention as used herein.

Polynucleotides or primers of the present invention may carry arevealing label. Suitable labels include radioisotopes such as ³²P or³⁵S, enzyme labels, or other labels such as biotin or digoxigenin. TheDIG™ system (Boehringer Mannheim) is useful as it offers a veryattractive non-radioactive system. The DIG system is based on thesteroid hapten digoxigenin, which occurs exclusively in Digitalis plantsand thus avoids endogenous background problems as in the case of otherhaptens, such as biotin} Such labels may be added to polynucleotides orprimers of the present invention and may be detected using by techniquesknown per se.

Polynucleotides such as a DNA polynucleotide and primers according tothe present invention may be produced recombinantly, synthetically, orby any means available to those of skill in the art. They may also becloned by standard techniques. In general, primers will be produced bysynthetic means, involving a step wise manufacture of the desirednucleic acid sequence one nucleotide at a time. Techniques foraccomplishing this using automated techniques are readily available inthe art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR cloning techniques. This will involvemaking a pair of primers (e.g. of about 15–30 nucleotides) to a regionof the nucleotide sequence which it is desired to clone, bringing theprimers into contact with mRNA or cDNA obtained from a fungal, plant orprokaryotic cell, performing a polymerase chain reaction underconditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

The nucleotide sequence of the present invention may be engineered inorder to alter its activity for a number of reasons, including but notlimited to, alterations which modify the processing and/or expression.For example, mutations may be introduced using techniques which are wellknown in the art, e.g., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference. By way of further example, the nucleotide sequence of thepresent invention may also be modified to optimise expression in aparticular host cell—such as the inclusion of additional promoter(s) orparts thereof, such as the provision of tandem repeats, and/or theprovision of other expression elements such as the intron sequence.Other sequence changes may be desired in order to introduce restrictionenzyme recognition sites.

Variant, Homologue and Fragment of the Promoter Nucleotide Sequence

The terms “variant”, “homologue” or “fragment” in relation to thenucleotide sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence has the ability to act as a promoter,preferably being at least as biologically active as the promoter havingthe sequence shown as SEQ ID No. 1. In particular, the term “homologue”covers homology with respect to structure and/or function providing theresultant nucleotide sequence has the ability to act as a promoter. Withrespect to sequence homology, preferably there is at least 75%, morepreferably at least 85%, more preferably at least 90% homology to thesequence shown as SEQ ID No. 1. More preferably there is at least 95%,more preferably at least 98%, homology to the sequence shown as SEQ IDNo. 1.

The present invention also relates to DNA segments comprising the DNAsequence of SEQ ID No. 1 or allelic variations of such sequences. Thesesegments are capable of acting as a regulatory region/unit.

Variant, Homologue and Fragment of Nucleotide Sequence SEQ ID No. 2

The terms “variant”, “homologue” or “fragment” in relation to thenucleotide sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence has the ability to increase expressionlevels, preferably being at least as biologically active as the sequenceshown as SEQ ID No. 2. In particular, the term “homologue” covershomology with respect to structure and/or function providing theresultant nucleotide sequence has the ability to increase expressionlevels. With respect to sequence homology, preferably there is at least75%, more preferably at least 85%, more preferably at least 90% homologyto the sequence shown as SEQ ID No. 2. More preferably there is at least95%, more preferably at least 98%, homology to the sequence shown as SEQID No. 2.

The present invention also relates to DNA segments comprising the DNAsequence of SEQ ID No. 2 or allelic variations of such sequences. Thesesegments are capable of increasing expression levels.

Variant, Homologue and Fragment of any One or More of the IdentifiedNucleotide Sequences in Table 1

The terms “variant”, “homologue” or “fragment” in relation to any one ofthe nucleotide sequences presented in Table 1 include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence has the ability to influenceexpression. In particular, the term “homologue” covers homology withrespect to structure and/or function providing the resultant nucleotidesequence has the ability to influence expression. With respect tosequence homology, preferably there is at least 75%, more preferably atleast 85%, more preferably at least 90% homology to the respectivesequence shown in Table 1. More preferably there is at least 95%, morepreferably at least 98%, homology to the respective sequence shown inTable 1.

NOI/POI

In a preferred aspect, the present invention relates to the use of thepromoter of the present invention to express one or more suitable NOIs.

Thus, in a preferred aspect, the promoter is operably linked to a NOI.

The term “operably linked” refers to a relationship—such as in asuitable juxtaposition—wherein the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a NOI is ligated in such a waythat expression of the coding sequence is achieved under conditioncompatible with the control sequences.

The NOI can encode for a POI.

The NOI can be any suitable sequence encoding a polypeptide of interest,other than the complete natural sequence normally associated with thepromoter of the present invention when the NOI is operably linked to thepromoter in their natural environment.

Preferably, the NOI does not include all of the nucleotide sequencenaturally associated with the wild type promoter of the presentinvention.

The NOI can be any nucleotide sequence that is either foreign(heterologous) or natural (homologous) to the organism in question—whichmay be a filamentous fungus or a plant.

Typically, the POI may be any suitable prokaryotic or eukaryoticheterologous or homologous peptide or protein of interest.

For some applications, the POI may be secreted and/or retrieved.

For some applications, it is important that the POI is secreted and/orretrieved.

For some applications, the POI may not secreted and/or retrieved.

For some applications, it is important that the POI is not secretedand/or retrieved.

The NOI may even be a sequence which is capable of expressing a nucleicacid, for example a regulatory RNA such as an antisense RNA or aribozyme, an mRNA, or a tRNA or rRNA capable of regulating themetabolism of an organism.

The NOI may also be a homologous nucleotide sequence which has beenmutated, such as by insertion, addition, deletion or alteration, suchthat it is no longer identical with the natural homologous nucleotidesequence.

The POI can be a single-chain polypeptide molecule as well as amultiple-polypeptide complex where individual constituent polypeptidesare linked by covalent or non-covalent means. Here, the term“polypeptide” includes peptides of two or more amino acids in length,typically having more than 5, or more than 10 or more than 20 aminoacids.

Typical examples of a NOI include sequences coding for proteins andenzymes that modify metabolic and catabolic processes. The heterologousnucleotide sequence may code for an agent for introducing or increasingpathogen resistance. The heterologous nucleotide sequence may be anantisense construct for modifying the expression of natural transcriptspresent in the relevant tissues.

The heterologous nucleotide sequence may be a protein giving nutritionalvalue to a food or crop. Typical examples include plant proteins thatcan aid human or animal digestion, inhibit the formation ofanti-nutritive factors and those plant proteins that have a moredesirable amino acid composition (e.g. a higher lysine content than anon-transgenic plant).

Non-limiting examples of POIs include, for example, proteins involved inthe regulation of cell division, for example growth factors includingneurotrophic growth factors, cytokines (such as α-, β- or γ-interferon,interleukins including IL-1, IL-2, tumour necrosis factor, orinsulin-like growth factors I or II), protein kinases (such as MAPkinase), protein phosphatases and cellular receptors for any of theabove.

The POI may also be an enzyme involved in cellular metabolic pathways,for example enzymes involved in carbohydrate biosynthesis ordegradation, amino acid biosynthesis or degradation (such as tyrosinehydroxylase), purine or pyrimidine biosynthesis or degradation, and thebiosynthesis or degradation of neurotransmitters, such as dopamine, or aprotein involved in the regulation of such pathways, for example proteinkinases and phosphatases.

The POI may also be effective in the post-harvest processing ofplants—e.g. in the brewing or baking processes.

The POI may also be a transcription factors or proteins involved intheir regulation, for example pocket proteins of the Rb family such asRb or p107, membrane proteins, structural proteins or heat shockproteins such as hsp70.

The NOI may code for an intron of a particular nucleotide sequence,wherein the intron can be in sense or antisense orientation.

Non-limiting examples of POIs include: proteins or enzymes involved instarch metabolism, proteins or enzymes involved in glycogen metabolism,acetyl esterases, aminopeptidases, amylases, arabinases,arabinofuranosidases, carboxypeptidases, catalases, cellulases,chitinases, chymosin, cutinase, deoxyribonucleases, epimerases,esterases, α-galactosidases, β-galactosidases, α-glucanases, glucanlysases, endo-β-glucanases, glucoamylases, glucose oxidases,α-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexoseoxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases,mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectin depolymerases, pectin methyl esterases, pectinolyticenzymes, peroxidases, phenoloxidases, phytases, polygalacturonases,proteases, rhamno-galacturonases, ribonucleases, thaumatin,transferases, transport proteins, transglutaminases, xylanases, orcombinations thereof. The NOI may even be an antisense sequence for anyof those sequences.

The NOI can be the nucleotide sequence coding for the exo-amylase enzymewhich is the subject of PCT patent application PCT/IB99/00649(incorporated herein by reference).

The NOI can be the nucleotide sequence coding for the xylanase enzymesand mutants thereof which are the subject of UK patent application GB99078057 (incorporated herein by reference).

The NOI can be the nucleotide sequence coding for thearabinofuranosidase enzyme which is the subject of PCT patentapplication PCT/EP96/01009 (incorporated herein by reference).

The NOI can be any of the nucleotide sequences coding for theADP-glucose pyrophosphorylase enzymes which are the subject of PCTpatent application PCT/EP94/01082 (incorporated herein by reference).

The NOI can be any of the nucleotide sequences coding for the α-glucanlyase enzyme which are described in PCT patent applicationPCT/EP94/03397 (incorporated herein by reference).

The NOI can be any of the sequences coding for T. lanuginosus amylase,as described in PCT patent application PCT/EP95/02607, incorporatedherein by reference.

The NOI can be any of the nucleotide sequences coding for the glucanaseenzyme which are described in PCT patent application PCT/EP96/01008(incorporated herein by reference).

The NOI can be any of the nucleotide sequences coding for theUDP-galactose epimerase enzyme, as well as anti-sense sequencestherefor,—such as those which are described in PCT patent applicationWO-A-98/54335 (incorporated herein by reference).

The NOI can be hox from the red algae Chondrus crispus or lipA fromAspergillus niger.

The POI can be a PME as disclosed in WO-A-97/03574 or the PME disclosedin either WO-A-94/25575 or WO-A-97/31102 as well as variants,derivatives or homologues of the sequences disclosed in those patentapplications.

The POI may even be a fusion protein, for example to aid in extractionand purification.

Examples of fusion protein partners include the maltose binding protein,glutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and β-galactosidase. It may also beconvenient to include a proteolytic cleavage site between the fusioncomponents.

The POI may even be fused to a secretion sequence. Examples of secretionleader sequences are those originating from the amyloglucosidase gene,the α-factor gene, the α-amylase gene, the lipase A gene, the xylanase Agene.

Other sequences can also facilitate secretion or increase the yield ofsecreted POI. Such sequences could code for chaperone proteins as forexample the product of Aspergillus niger cyp B gene described in UKpatent application 9821198.0.

The NOI may be engineered in order to alter their activity for a numberof reasons, including but not limited to, alterations which modify theprocessing and/or expression of the expression product thereof. Forexample, mutations may be introduced using techniques which are wellknown in the art, e.g., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference. By way of further example, the NOI may also be modified tooptimise expression in a particular host cell. Other sequence changesmay be desired in order to introduce restriction enzyme recognitionsites.

The NOI may include within it synthetic or modified nucleotides. Anumber of different types of modification to oligonucleotides are knownin the art. These include methylphosphonate and phosphorothioatebackbones, addition of acridine or polylysine chains at the 3′ and/or 5′ends of the molecule. For the purposes of the present invention, it isto be understood that the NOI may be modified by any method available inthe art. Such modifications may be carried out in to enhance the in vivoactivity or life span of the NOI.

The NOI may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences of the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes the nucleotide sequence according tothe present invention directly or indirectly attached to a NOI. Anexample of an indirect attachment is the provision of a suitable spacergroup such as an intron sequence, such as the Sh1-intron or the ADHintron, intermediate the promoter and the NOI. The same is true for theterm “fused” in relation to the present invention which includes director indirect attachment. In each case, the terms do not cover the naturalcombination of the wild type gene promoter and its associated nucleotidesequence when they are both in their natural environment.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a plant cell intowhich it has been transferred.

The selectable marker means may reside on an additional vector or may beincluded in the nucleic acid molecule which contains the expressionsystem. The nature of the selectable marker means will depend on thenature of the host and the culture conditions. The most suitableselection systems for industrial micro-organisms are those formed by thegroup of selection markers which do not require a mutation in the hostorganism.

U.S. Pat. No. 5,358,864 provides a short list of suitable selectablemarker genes that may be used in the present invention—examples of whichinclude fungal selection markers such as those that are the genes foracetamidase (amdS), ATP synthetase, subunit 9 (oliC) and benomylresistance (benA).

Examples of non-fungal selection markers are the bacterial G418resistance gene (this may also be used in yeast, but not in fungi), theampicillin resistance gene (E. coli), the neomycin resistance gene(Bacillus) and the E. coli uidA gene, coding for β-glucuronidase (GUS).

In certain aspects of the present invention, use of the ble marker—whichconfers resistance to phleomycin/bleomycin/zeocin—may be preferred.However, other selection markers, known in the art, could be used.Examples of auxotrophic markers are pyrG selecting for uridineprototrophs, argB selecting for arginine prototrophs, niaD selecting fornitrate prototrophs, trpC selecting for tryptophan prototrophs, amdSselecting for increased utilisation of acetamide as sole nitrogensource. Dominant resistance markers could be chosen from oliC3conferring resistance to oligomycin, hph conferring resistance tohygromycin B, bar conferring resistance to bialaphos or NPTII conferringresistance to G418.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a bacterium,preferably of the genus Bacillus, such as Bacillus subtilis, or plants,such as potatoes, sugar beet etc., into which it has been transferred.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a plant. Variousmarkers exist which may be used, such as for example those encodingmannose 6-phosphate isomerase, glucosamine 6-phosphatedeaminase/ketoisomerase, xylose isomerase, or those markers that providefor antibiotic resistance—e.g. resistance to G418, hygromycin,bleomycin, kanamycin and gentamycin.

Vectors

The term “vector” includes expression vectors, replicable vectors,transformation vectors and shuttle vectors, including vectorcombinations thereof.

The term “expression vector” means a construct capable of in vivo or invitro expression.

Preferably the expression vector is incorporated in the genome of theorganism. The term “incorporated” preferably covers stable incorporationinto the genome.

Preferably, the vector of the present invention comprises a constructaccording to the present invention. Alternatively expressed, preferablythe promoter of the invention is present in a vector and wherein thepromoter is operably linked to a NOI such that the promoter is capableof providing for the expression of the coding sequence by a suitablehost organism, i.e. the vector is an expression vector.

The term “transformation vector” means a construct capable of beingtransferred from one entity to another entity—which may be of thespecies or may be of a different species. If the construct is capable ofbeing transferred from one species to another—such as from an E. coliplasmid to a bacterium, such as of the genus Bacillus, then thetransformation vector is sometimes called a “shuttle vector”. It mayeven be a construct capable of being transferred from an E. coli plasmidto an Agrobacterium to a plant.

The vectors of the present invention may be transformed into a suitablehost cell to provide for expression of a POI.

Thus, in a further aspect the invention provides a process for preparinga POI invention which comprises cultivating a host cell transformed ortransfected with an expression vector under conditions to provide forexpression by the promoter of the present invention of a NOI encodingthe POI, and optionally recovering the expressed POI.

Typically, the nucleotide sequences of the invention may be incorporatedinto a recombinant replicable vector. The vector may be used toreplicate the nucleic acid in a compatible host organism.

Thus, in a further embodiment, the invention provides a method ofintroducing a nucleotide sequence of the invention into a replicablevector, introducing the vector into a compatible host organism, andgrowing the host organism under conditions which bring about expressionof a NOI by the promoter of the present invention. The POI may then berecovered from the host organism. Suitable host organisms include plantsor plant cells.

The NOI may be incorporated into a replicable vector, for example acloning or expression vector, which comprises a promoter of the presentinvention. The vector may be used to replicate the NOI in a compatiblehost cell.

Thus, in a further embodiment, the invention provides a method of makingNOIs by introducing a NOI into a replicable vector which comprises thepromoter of the present invention, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector. The vector may be recovered fromthe host cell.

The vectors may be for example, plasmid, virus or phage vectors. Inaddition to the promoter of the present invention, the vectors may beprovided with any one or more of an origin of replication, a NOIoperably linked to the promoter for the expression of the NOI, and aregulator of the promoter.

Vectors may be used, for example, to transfect or transform a hostorganism either in vitro or in vivo.

Such vectors may be transformed or transfected into a suitable hostorganism to provide for expression of a protein of the invention. Thisprocess may comprise culturing a host organism transformed with anexpression vector as described above under conditions to provide forexpression by the vector of a coding sequence encoding the protein, andoptionally recovering the expressed protein.

Vectors of the invention may be introduced into host organisms for thepurpose of replicating the vectors/nucleotide sequences and/orexpressing the NOI. In one preferred aspect, the host organism is aplant cell.

Vectors of the present invention may introduced into suitable hostorganisms using a variety of techniques known in the art, such astransfection, transformation and electroporation. Another technique isthe protoplast transformation method (Winer et al., Microbiology, 1985,468, American Society for Microbiology).

Vectors may be used in vitro, for example for the production of RNA orused to transfect or transform a host cell.

Tissue

The term “tissue” as used herein includes tissue per se and organ.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that could comprise the promoter of the present invention and/orproducts obtained therefrom, wherein the promoter can allow expressionof a NOI when present in the host cell.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a promoter of the present invention.Preferably the promoter is carried in a vector for the replication andexpression of NOIs. The cells will be chosen to be compatible with thesaid vector and may for example be prokaryotic (for example bacterial),fungal, yeast or plant cells.

Preferably, the host cell is a plant cell.

The gram-negative bacterium E. coli is widely used as a host forheterologous gene expression. However, large amounts of heterologousprotein tend to accumulate inside the cell. Subsequent purification ofthe desired protein from the bulk of E. coli intracellular proteins cansometimes be difficult.

In contrast to E. coli, bacteria from the genus Bacillus are verysuitable as heterologous hosts because of their capability to secreteproteins into the culture medium. Other bacteria suitable as hosts arethose from the genera Streptomyces and Pseudomonas.

Depending on the nature of the NOI encoding the POI, and/or thedesirability for further processing of the expressed protein, eukaryotichosts such as yeasts or fungi may be preferred. In general, yeast cellsare preferred over fungal cells because they are easier to manipulate.However, some proteins are either poorly secreted from the yeast cell,or in some cases are not processed properly (e.g. hyperglycosylation inyeast). In these instances, a fungal host organism should be selected.

Examples of suitable expression hosts within the scope of the presentinvention are fungi such as Aspergillus species (such as those describedin EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria suchas Bacillus species (such as those described in EP-A-0134048 andEP-A-0253455), Streptomyces species and Pseudomonas species; and yeastssuch as Kluyveromyces species (such as those described in EP-A-0096430and EP-A-0301670) and Saccharomyces species. By way of example, typicalexpression hosts may be selected from Pichia pastoris, Hansenulapolymorpha, Aspergillus niger, Aspergillus niger var. tubigenis,Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillusnidulans, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactisand Saccharomyces cerevisiae.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

Thus, the present invention also provides a method of transforming ahost cell with a nucleotide sequence shown as SEQ ID No. 1 or aderivative, homologue, variant or fragment thereof.

Host cells transformed with a promoter according to the presentinvention operably linked to a NOI may be cultured under conditionssuitable for the expression and recovery of the POI from cell culture.The POI may be secreted or may be contained intracellularly depending onthe sequence and/or the vector used. As will be understood by those ofskill in the art, expression vectors containing a promoter according tothe present invention operably linked to a NOI can be designed withsignal sequences which direct secretion of the NOI/POI through aparticular prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may join the NOI to a nucleotide sequence encoding apolypeptide domain which will facilitate purification of solubleproteins (Kroll D J et al (1993) DNA Cell Biol 12:441–53).

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence of the presentinvention, wherein the promoter can allow expression of a NOI whenpresent in the organism. Examples of organisms may include a fungus,yeast or a plant.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence of thepresent invention, wherein the promoter can allow expression of a NOIwithin the organism. Preferably the nucleotide sequence is incorporatedin the genome of the organism.

The term “transgenic organism” does not cover the native nucleotidesequence according to the present invention in its natural environmentwhen it is operably linked to its associated coding sequence which isalso in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence of the present invention, constructs according to the presentinvention (including combinations thereof), vectors according to thepresent invention, plasmids according to the present invention, cellsaccording to the present invention, tissues according to the presentinvention or the products thereof. The transformed cell or organismcould prepare acceptable quantities of the POI. In some instances, thePOI may be easily retrievable from, the cell or organism.

Transformation of Host Cells/Host Organisms

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis. Teachings on the transformation ofprokaryotic hosts is well documented in the art, for example seeSambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., CurrentProtocols in Molecular Biology (1995), John Wiley & Sons, Inc.

If a prokaryotic host is used then the NOI may need to be suitablymodified before transformation—such as by removal of introns.

Transformed Fungus

A host organism may be a fungus—such as a mold. Examples of suitablesuch hosts include any member belonging to the genera Thermomyces,Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma andthe like—such as Thermomyces lanuginosis, Acremonium chrysogenum,Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillinumchrysogenem, Mucor javanious, Neurospora crassa, Trichoderma viridae andthe like.

In one embodiment, the host organism may be a filamentous fungus.

For almost a century, filamentous fungi have been widely used in manytypes of industry for the production of organic compounds and enzymes.For example, traditional Japanese koji and soy fermentations have usedAspergillus sp. Also, in this century Aspergillus niger has been usedfor production of organic acids particular citric acid and forproduction of various enzymes for use in industry.

There are two major reasons why filamentous fungi have been so widelyused in industry. First filamentous fungi can produce high amounts ofextracellular products, for example enzymes and organic compounds suchas antibiotics or organic acids. Second filamentous fungi can grow onlow cost substrates such as grains, bran, beet pulp etc. The samereasons have made filamentous fungi attractive organisms as hosts forheterologous expression according to the present invention.

In order to prepare the transgenic Aspergillus, expression constructsare prepared by inserting the nucleotide sequence according to thepresent invention (and optionally the NOI) into a construct designed forexpression in filamentous fungi.

Several types of constructs used for heterologous expression have beendeveloped. These constructs preferably contain one or more of: a signalsequence which directs the POI to be secreted, typically being of fungalorigin, and a terminator (typically being active in fungi) which endsthe expression system.

Another type of expression system has been developed in fungi where thenucleotide sequence according to the present invention (and optionallythe NOI) can be fused to a smaller or a larger part of a fungal geneencoding a stable protein. This can stabilise the POI. In such a systema cleavage site, recognised by a specific protease, can be introducedbetween the fungal protein and the POI, so the produced fusion proteincan be cleaved at this position by the specific protease thus liberatingthe POI. By way of example, one can introduce a site which is recognisedby a KEX-2 like peptidase found in at least some Aspergilli. Such afusion leads to cleavage in vivo resulting in production of theexpressed product and not a larger fusion protein.

Heterologous expression in Aspergillus has been reported for severalgenes coding for bacterial, fungal, vertebrate and plant proteins. Theproteins can be deposited intracellularly if the nucleotide sequenceaccording to the present invention (or even the NOI) is not fused to asignal sequence. Such proteins will accumulate in the cytoplasm and willusually not be glycosylated which can be an advantage for some bacterialproteins. If the nucleotide sequence according to the present invention(or even the NOI) is equipped with a signal sequence the protein willaccumulate extracellularly.

With regard to product stability and host strain modifications, someheterologous proteins are not very stable when they are secreted intothe culture fluid of fungi. Most fungi produce several extracellularproteases which degrade heterologous proteins. To avoid this problemspecial fungal strains with reduced protease production have been usedas host for heterologous production.

Teachings on transforming filamentous fungi are reviewed in U.S. Pat.No. 5,741,665 which states that standard techniques for transformationof filamentous fungi and culturing the fungi are well known in the art.An extensive review of techniques as applied to N. crassa is found, forexample in Davis and de Serres, Methods Enzymol (1971) 17A:79–143.Standard procedures are generally used for the maintenance of strainsand the preparation of conidia. Mycelia are typically grown in liquidcultures for about 14 hours (25° C.), as described in Lambowitz et al.,J Cell Biol (1979) 82:17–31. Host strains can generally be grown ineither Vogel's or Fries minimal medium supplemented with the appropriatenutrient(s), such as, for example, any one or more of: his, arg, phe,tyr, trp, p-aminobenzoic acid, and inositol.

Further teachings on transforming filamentous fungi are reviewed in U.S.Pat. No. 5,674,707 which states that once a construct has been obtained,it can be introduced either in linear form or in plasmid form, e.g., ina pUC-based or other vector, into a selected filamentous fungal hostusing a technique such as DNA-mediated transformation, electroporation,particle gun bombardment, protoplast fusion and the like. In addition,Ballance 1991 (ibid) states that transformation protocols for preparingtransformed fungi are based on preparation of protoplasts andintroduction of DNA into the protoplasts using PEG and Ca²⁺ ions. Thetransformed protoplasts then regenerate and the transformed fungi areselected using various selective markers.

To allow for selection of the resulting transformants, thetransformation typically also involves a selectable gene marker which isintroduced with the expression cassette, either on the same vector or byco-transformation, into a host strain in which the gene marker isselectable. Various marker/host systems are available, including thepyrG, argB and niaD genes for use with auxotrophic strains ofAspergillus nidulans; pyrG and argB genes for Aspergillus oryzaeauxotrophs; pyrG, trpC and niaD genes for Penicillium chrysogenumauxotrophs; and the argB gene for Trichoderma reesei auxotrophs.Dominant selectable markers including amdS, oliC, hyg and phleo are alsonow available for use with such filamentous fungi as A. niger, A.oryzae, A. ficuum, P. chrysogenum, Cephalosporium acremonium,Cochliobolus heterostrophus, Glomerella cingulata, Fulvia fulva andLeptosphaeria maculans (for a review see Ward in Modern MicrobialGenetics, 1991, Wiley-Liss, Inc., at pages 455–495). A commonly usedtransformation marker is the amdS gene of A. nidulans which in high copynumber allows the fungus to grow with acrylamide as the sole nitrogensource.

For the transformation of filamentous fungi, several transformationprotocols have been developed for many filamentous. Among the markersused for transformation are a number of auxotrophic markers such asargB, trpc, niaD and pyrG, antibiotic resistance markers such as benomylresistance, hygromycin resistance and phleomycin resistance.

In one aspect, the host organism can be of the genus Aspergillus, suchas Aspergillus niger.

A transgenic Aspergillus according to the present invention can also beprepared by following the teachings of Rambosek, J. and Leach, J. 1987(Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit.Rev. Biotechnol. 6:357–393), Davis R. W. 1994 (Heterologous geneexpression and protein secretion in Aspergillus. In: Martinelli S. D.,Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress inindustrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525–560),Ballance, D. J. 1991 (Transformation systems for Filamentous Fungi andan Overview of Fungal Gene structure. In: Leong, S. A., Berka R. M.(Editors) Molecular Industrial Mycology. Systems and Applications forFilamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1–29) and TurnerG. 1994 (Vectors for genetic manipulation. In: Martinelli S. D.,Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress inindustrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641–666).

Transformed Yeast

In another embodiment the transgenic organism can be a yeast.

In this regard, yeast have also been widely used as a vehicle forheterologous gene expression.

By way of example, the species Saccharomyces cerevisiae has a longhistory of industrial use, including its use for heterologous geneexpression. Expression of heterologous genes in Saccharomyces cerevisiaehas been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berryet al, eds, pp 401–429, Allen and Unwin, London) and by King et al(1989, Molecular and Cell Biology of Yeasts, E F Walton and G TYarronton, eds, pp 107–133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrativevectors, which require recombination with the host genome for theirmaintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructsare prepared by inserting the nucleotide sequence of the presentinvention into a construct designed for expression in yeast. Severaltypes of constructs used for heterologous expression have beendeveloped. The constructs may contain a promoter active in yeast, suchas a promoter of yeast origin, such as the GALL promoter, is used.Usually a signal sequence of yeast origin, such as the sequence encodingthe SUC2 signal peptide, is used. A terminator active in yeast ends theexpression system.

For the transformation of yeast several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al (1978, Proceedings of the National Academy of Sciences of the USA75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al(1983, J Bacteriology 153, 163–168).

The transformed yeast cells may be selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, eg G418.

Transformed Plants/Plant Cells

A preferred host organism suitable for the present invention is a plant.

In this respect, the basic principle in the construction of geneticallymodified plants is to insert genetic information in the plant genome soas to obtain a stable maintenance of the inserted genetic material.

Several techniques exist for inserting the genetic information, the twomain principles being direct introduction of the genetic information andintroduction of the genetic information by use of a vector system. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205–225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17–27).

Even though the promoter of the present invention is not disclosed inEP-B-0470145 and CA-A-2006454, those two documents do provide someuseful background commentary on the types of techniques that may beemployed to prepare transgenic plants according to the presentinvention. Some of these background teachings are now included in thefollowing commentary.

The basic principle in the construction of genetically modified plantsis to insert genetic information in the plant genome so as to obtain astable maintenance of the inserted genetic material.

Thus, in one aspect, the present invention relates to a vector systemwhich carries a nucleotide sequence or construct according to thepresent invention and which is capable of introducing the nucleotidesequence or construct into the genome of an organism, such as a plant.

The vector system may comprise one vector, but it can comprise twovectors. In the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1–19.

One extensively employed system for transformation of plant cells with agiven promoter or nucleotide sequence or construct is based on the useof a Ti plasmid from Agrobacterium tumefaciens or a R1 plasmid fromAgrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301–305and Butcher D. N. et al. (1980), Tissue Culture Methods for PlantPathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203–208.

Several different Ti and Ri plasmids have been constructed which aresuitable for the construction of the plant or plant cell constructsdescribed above. A non-limiting example of such a Ti plasmid is pGV3850.

The nucleotide sequence or construct of the present invention shouldpreferably be inserted into the Ti-plasmid between the terminalsequences of the T-DNA or adjacent a T-DNA sequence so as to avoiddisruption of the sequences immediately surrounding the T-DNA borders,as at least one of these regions appear to be essential for insertion ofmodified T-DNA into the plant genome.

As will be understood from the above explanation, if the organism is aplant, then the vector system of the present invention is preferably onewhich contains the sequences necessary to infect the plant (e.g. the virregion) and at least one border part of a T-DNA sequence, the borderpart being located on the same vector as the genetic construct.Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmidor an Agrobacterium rhizogenes R1-plasmid or a derivative thereof, asthese plasmids are well-known and widely employed in the construction oftransgenic plants, many vector systems exist which are based on theseplasmids or derivatives thereof.

In the construction of a transgenic plant the nucleotide sequence orconstruct of the present invention may be first constructed in amicro-organism in which the vector can replicate and which is easy tomanipulate before insertion into the plant. An example of a usefulmicro-organism is E. coli., but other micro-organisms having the aboveproperties may be used. When a vector of a vector system as definedabove has been constructed in E. coli. it is transferred, if necessary,into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.The Ti-plasmid harbouring the nucleotide sequence or construct of theinvention is thus preferably transferred into a suitable Agrobacteriumstrain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cellharbouring the nucleotide sequence or construct of the invention, whichDNA is subsequently transferred into the plant cell to be modified.

As reported in CA-A-2006454, a large amount of cloning vectors areavailable which contain a replication system in E. coli and a markerwhich allows a selection of the transformed cells. The vectors containfor example pBR 322, the pUC series, the M13 mp series, pACYC 184 etc.

In this way, the nucleotide or construct of the present invention can beintroduced into a suitable restriction position in the vector. Thecontained plasmid is used for the transformation in E. coli. The E. colicells are cultivated in a suitable nutrient medium and then harvestedand lysed. The plasmid is then recovered. As a method of analysis thereis generally used sequence analysis, restriction analysis,electrophoresis and further biochemical-molecular biological methods.After each manipulation, the used DNA sequence can be restricted andconnected with the next DNA sequence. Each sequence can be cloned in thesame or different plasmid.

After each introduction method of the desired promoter or construct ornucleotide sequence according to the present invention in the plants thepresence and/or insertion of further DNA sequences may be necessary. If,for example, for the transformation the Ti- or Ri-plasmid of the plantcells is used, at least the right boundary and often however the rightand the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areasof the introduced genes, can be connected. The use of T-DNA for thetransformation of plant cells has been intensively studied and isdescribed in EP-A-120516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley,et al., Crit. Rev. Plant Sci., 4:1–46; and An et al., EMBO J. (1985)4:277–284.

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson, 203–208. For further teachings on thistopic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]42:205–225) and Christou (Agro-Food-Industry Hi-Tech Mar./Apr. 199417–27). With this technique, infection of a plant may be done on acertain part or tissue of the plant, i.e. on a part of a leaf, a root, astem or another part of the plant.

Typically, with direct infection of plant tissues by Agrobacteriumcarrying the promoter and/or the GOI, a plant to be infected is wounded,e.g. by cutting the plant with a razor or puncturing the plant with aneedle or rubbing the plant with an abrasive. The wound is theninoculated with the Agrobacterium. The inoculated plant or plant part isthen grown on a suitable culture medium and allowed to develop intomature plants.

When plant cells are constructed, these cells may be grown andmaintained in accordance with well-known tissue culturing methods suchas by culturing the cells in a suitable culture medium supplied with thenecessary growth factors such as amino acids, plant hormones, vitamins,etc. Regeneration of the transformed cells into genetically modifiedplants may be accomplished using known methods for the regeneration ofplants from cell or tissue cultures, for example by selectingtransformed shoots using an antibiotic and by subculturing the shoots ona medium containing the appropriate nutrients, plant hormones, etc.

Other techniques for transforming plants include ballistictransformation, the silicon whisker carbide technique (see Frame B R,Drayton P R, Bagnaall S V, Lewnau C J, Bullock W P, Wilson H M, DunwellJ M, Thompson J A & Wang K (1994) Production of fertile transgenic maizeplants by silicon carbide whisker-mediated transformation, The PlantJournal 6: 941–948) and viral transformation techniques (e.g. see MeyerP, Heidmann I & Niedenhof I (1992) The use of cassaya mosaic virus as avector system for plants, Gene 110: 213–217).

Further teachings on plant transformation may be found in EP-A-0449375.

Ballistic Transformation of Plants and Plant Tissue

Originally developed to produce stable transformants of plant specieswhich were recalcitrant to transformation by Agrobacterium tumefaciens,ballistic transformation of plant tissue, which introduces DNA intocells on the surface of metal particles, has found utility in testingthe performance of genetic constructs during transient expression. Inthis way, gene expression can be studied in transiently transformedcells, without stable integration of the gene in interest, and therebywithout time-consuming generation of stable transformants.

In more detail, the ballistic transformation technique (otherwise knownas the particle bombardment technique) was first described by Klein etal. [1987], Sanford et al. [1987] and Klein et al. [1988] and has becomewidespread due to easy handling and the lack of pre-treatment of thecells or tissue in interest.

The principle of the particle bombardment technique is direct deliveryof DNA-coated micro-projectiles into intact plant cells by a drivingforce (e.g. electrical discharge or compressed air). Themicro-projectiles penetrate the cell wall and membrane, with only minordamage, and the transformed cells then express the promoter constructs.

One particle bombardment technique that can be performed uses theParticle Inflow Gun (PIG), which was developed and described by Finer etal. [1992] and Vain et al. [1993]. The PIG accelerates themicro-projectiles in a stream of flowing helium, through a partialvacuum, into the plant cells.

One of advantages of the PIG is that the acceleration of themicro-projectiles can be controlled by a timer-relay solenoid and byregulation the provided helium pressure. The use of pressurised heliumas a driving force has the advantage of being inert, leaves no residuesand gives reproducible acceleration. The vacuum reduces the drag on theparticles and lessens tissue damage by dispersion of the helium gasprior to impact [Finer et al. 1992].

In some cases, the effectiveness and ease of the PIG system makes it agood choice for the generation of transient transformed guar tissue,which were tested for transient expression of promoter/reporter genefusions.

Guar

As indicated above, in one aspect, a preferred transformed organism istransformed guar.

Guar (Cyamopsis tetragonoloba) is a drought-tolerant species, whichoriginated in India and Pakistan, but is cultivated for industrial usein a number of other countries e.g. USA. Guar is taxonomically arrangedwith the peas and beans in the family of grain legumes. Legumes aredicotyledonous plants (dicots), which are characterised by having broadleaves and two cotyledons, in contrast to monocotyledonous plants(monocots) which display ‘grass like’ morphology and only have one.Examples of other dicots are tobacco and potato, and examples ofmonocots are cereals such as wheat, rice, maize and barley. Guar differsfrom most crop legumes such as peas and beans, however, in that its seedcarbohydrate reserve is not starch in the cotyledons, but isgalactomannan accumulated in the endosperm. The endosperm is anon-photosynthetic tissue, which envelopes the cotyledons in the seedimmediately beneath the seed coat, and can be dissected away from theother seed tissues.

Guar Gum

Guar is grown as crop for animal feed and human consumption. However, animportant use of guar is the extraction of guar gum or ‘guaran’, whichis used as a functional ingredient in foods e.g. as thickener in icecream. Guar gum consists of galactomannan—which is an extracellularcell-wall polysaccharide located in the endosperm.

In a preferred aspect, the present invention relates to the use of thepromoter of the present invention to cause expression of an NOI that canaffect guar gum synthesis.

Galactomannan

Galactomannan consists of a mannan backbone with substitutedgalactosyl-groups, the bonding of which is(1→6)-α-D-galacto-(1→4)-β-D-mannan, and the polymer has an molecularweight of 220,000 daltons (Whistler & Hymowitz 1979).

The degree of substitution, and thereby the ratio between galactose andmannose residues in galactomannan, varies between leguminous species.The ratio is approx. 0.56 in guar, and mature seeds contains approx.35–42% galactomannan (Whistler & Hymowitz 1979).

In a preferred aspect, the present invention relates to the use of thepromoter of the present invention to cause expression of an NOI that canaffect galactomannan synthesis.

Endosperm Expression

In a preferred aspect, the promoter of the present invention is activein the endosperm of plant tissue.

In this respect, endosperm—as well as the embryo—are formed early inseed development of flowering plants. In particular, the endosperm isformed during double fertilisation in which, one sperm nucleus fuseswith an egg to produce the embryo, and a second sperm nucleus fuses withtwo polar nuclei to form the triploid endosperm [Lopes & Larkin 1993].In some, but by no means all, plants this tissue serves as a storagereservoir for the seeds, and guar and the cereals are among these. Inother species, however, the cotyledons have assumed the status of theprincipal storage tissue of the seed, and the endosperm has becomevestigial.

In cereals such as rice, wheat, barley, and maize the chief carbohydratereserve in the endosperm is starch which accumulates intracellularly inplastids known as amyloplasts. In guar endosperm, however, the principalstorage carbohydrate is galactomannan which accumulates extracellularlyin the intercellular spaces, and is therefore secreted out of the cell.

During maturation, the developing seed increases in volume and mass dueto significant cell expansion and accumulation of carbohydrates, proteinand lipids to be used as C and N sources during germination. Aftermaturation, the seed enters dormancy [West & Harada 1993], imposed byplant hormones, such as abscisic acid (ABA), which prevent precociousgermination [Thomas 1993]. On germination of guar seeds, mannanase, andgalactosidase activities break down the galactomannan of the endosperm,and the mannose and galactose released support the development of theseedling.

Carbohydrate Metabolism

In a preferred aspect, the present invention relates to the use of thepromoter of the present invention to cause expression of an NOI that canaffect carbohydrate metabolism, such as sucrose metabolism, particularlyin a plant tissue.

In this respect, sucrose is the major photoassimilate which istransported long distances in plants from the photosynthetic ‘source’tissue to the heterotrophic energy-consuming ‘sink’ tissues, in which itis the mainstay of carbohydrate nutrition.

The initial precursor for the synthesis of sucrose is triose-phosphatewhich is synthesised in the carbon-fixation (Calvin) cycle inchloroplast in photosynthetic cells. This triose phosphate(glyceraldehyde 3-phosphate) is transported to the cytoplasm andconverted to fructose 6-phosphate (Fructose-6-P) and glucose 1-phosphate(Glucose-1-P). The Glucose-1-P is converted to UDP-glucose whichtogether with Fructose-6-P are converted to sucrose 6-phosphate(Sucrose-6-P) by sucrose phosphate synthase. S-6-P is the immediateprecursor for sucrose and the conversion is catalysed by sucrosephosphatase.

After its synthesis in the cytoplasm of mesophyll cells, sucrose isloaded by the companion cells into the phloem and distributed to sinktissues. The transport process is a ‘mass flow’ facilitated by osmoticpressure, due to a concentration gradient of sucrose between source andsink. Photosynthesis is slowed by build up of sucrose in green tissues,and so it is seen by many as a demand-led process, with the vasculartissue communicating requirement for carbohydrate by unloading ofsucrose into developing sink tissues.

The developing seed is a storage-active sink organ, and is therefore alarge user of sucrose. Developing seeds utilise sucrose for severalpurposes, one example of which is synthesis of storage polysaccharidesas galactomannan in guar endosperm.

Sucrose is degraded by sucrose synthase to fructose (F) andUDP-galactose (UDPG) and through a series of conversions GDP-mannose(GDPM) and UDPG are formed. GDPM and UDPG are the immediate precursorsfor galactomannan and the synthesis is catalysed by mannan synthase andgalactosyl transferase. Galactomannan is synthesized and transportedfrom the golgi apparatus to the extracellular space. Another key enzymein the synthesis of galactomannan is UDP galactose 4-epimerase.

Breakdown of sucrose in plants can be achieved through the activity oftwo enzymes: Invertase (E.C. 3.2.1.26) and sucrose synthase (E.C.2.4.1.13). The name ‘sucrose synthase’ implies that the enzyme catalysesthe synthesis of sucrose, but the metabolic role of sucrose synthase iscatabolic rather than anabolic, and sucrose synthase preferentiallycleaves sucrose in vivo. Consistent with this role, sucrose synthase isabundant in sink tissues such as developing seeds, but not in fullycompetent photosynthetic tissues. Sucrose synthase catalyses thecleavage of sucrose in the presence of UDP into UDP-glucose andfructose. By cleavage of sucrose in the presence of UDP the high energyof the sucrose glycosidic link is conserved in UDP-glucose, which canthen serve as a glycosidic donor for further reactions. Invertase, onthe other hand, catalyses the hydrolysis of sucrose into glucose andfructose, and probably serves less to provide carbohydrate for storage,but may furnish the immediate energy requirements of development.

In a preferred aspect, the present invention relates to the use of thepromoter of the present invention to cause expression of an NOI that canaffect the in situ activity of UDP galactose 4-epimerase.

Gus Reporter Gene

In some of the studies of the present invention, the E. coli uidA gene(otherwise known as the GUS gene) is used as a reporter gene. Here, theE. coli uidA gene is used as proof that the promoter of the presentinvention can cause expression of an NOI. However, it is to be notedthat the present invention is not limited to the NOI just being the E.coli uidA gene.

In more detail, the uidA gene codes for the β-glucuronidase enzyme (GUS)which is a hydrolase that catalyses the cleavage of β-glucuronides, e.g.X-gluc and MUG. The GUS reporter gene system for higher plants wasdeveloped by Jefferson 1987.

The system utilises that the uidA gene can be fused to a promoter andintroduced in to plant cells for expression. By assaying the activity ofthe GUS protein in transgenic tissue, the activity of a given promotercan be monitored. In some of our studies, the E. coli uidA gene has beenmodified to prevent glycosylation in plants, which enables GUS to retainactivity when targeted to the endoplasmatic reticulum (ER).

For transient expression studies, X-Gluc (5-bromo-4-chloro-3-indolylb-D-glucuronide) is used as a substrate in histochemical detection ofGUS (qualitative), and MUG (4-methyl umbelliferyl glucuronide) as asubstrate for fluorometrical detection (quantitative). The enzymaticcleavage of the colourless X-Gluc substrate produces a blue indigoprecipitate at the site of cleavage. The precipitation is caused byoxidative dimerisation of the indoxyl derivative produced by thecleavage of X-Gluc. The activity of GUS can therefore be detected andanalysed in transgenic tissue without extracting the enzyme beforerunning the assay.

Production of Poi

Host organisms comprising the vectors of the present invention may beused to express the POI by use of the promoter of the present invention.In this respect, host organisms may be cultured under suitableconditions which allow expression of the POI. In some instances,expression of the POI may be constitutive such that they are continuallyproduced, or inducible, requiring an inducer to initiate expression. Inthe case of inducible expression, protein production can be initiatedwhen required by, for example, addition of an inducer substance to theculture medium.

Once the vector has been transformed or transfected into a suitable hostorganism then the host organism can be cultivated. Here reference can bemade briefly to U.S. Pat. No. 5,543,322 which says that for cultivationof a transformant, a culture medium containing carbon and nitrogensources assimilable by the transformant and the like can be used. Anycarbon source assimilable by the transformant can be used. Examplesthereof include glucose, sucrose, starch, soluble starch, dextrin,glycerin, n-paraffin and the like as well as organic acids (e.g., aceticacid, fumaric acid, benzoic acid, etc.), alcohols (e.g., methanol,ethanol, butanol, etc.), fats and oils (soybean oil, lard, etc.) and thelike. They can be used alone or in combination thereof. As the nitrogensources, there are, for example, peptone, soybean flour, cotton seedflour, meat extract, yeast extract, dried yeast, corn steep liquor, corngluten meal, urea, ammonium salts (e.g., ammonium chloride, ammoniumsulfate, etc.), nitrates (e.g., potassium nitrate, ammonium nitrate,etc.), other organic or inorganic nitrogen-containing materials and thelike. They can be used alone or in combination thereof. In addition,inorganic salts (e.g., phosphates, etc.), trace metal salts (e.g.,magnesium salt, calcium salt, manganese salt, etc.) can be appropriatelyadded.

If desired, the POI can be extracted from host organisms by a variety oftechniques known in the art, including enzymatic, chemical and/orosmotic lysis and physical disruption. For some applications, apreferred extraction/purification protocol may involve a centriguationstep followed by, if necessary using, column chromatography such asion-exchange or affinity chromatography.

Thus, after the desired product (e.g. the POI) has accumulated in aculture medium, a supernatant fluid containing the POI can be obtainedby centrifugation or filtration. On the other hand, when the POI hasaccumulated in the organisms, after the cultivation, the organisms canbe collected by a known method and the desired product is recovered byan appropriate method. For example, the organisms can be suspended in abuffer containing a protein denaturant such as guanidine hydrochloride,the suspension is stirred in a cold place, and then the supernatantfluid containing the desired product is obtained by centrifugation orthe like.

Alternatively, after the organisms have been suspended in a buffer, theorganisms can be ground by glass beads, or broken by French press,sonication, enzymatic treatment or the like, and then the supernatantfluid is obtained by centrifugation or the like.

For separation and purification of the POI from the above supernatantfluid reference can be made to U.S. Pat. No. 554,332 where it is statedthat per se known separation and purification methods can beappropriately combined. As the known separation and purificationmethods, there are, for example, a method utilizing a difference insolubilities (e.g., salting out, precipitation with a solvent, etc.), amethod mainly utilizing a difference in molecular weights (e.g.,dialysis, ultrafiltration, gel filtration, etc.), a method utilizing adifference in charges (e.g., ion exchange chromategraphy, etc.), amethod utilizing specific affinity (e.g., affinity chromatography,etc.), a method utilizing a difference in hydrophobicities (e.g.,reverse phase high performance liquid chromatography, etc.), a methodutilizing a difference in isoelectric points (e.g., isoelectricfocusing, etc.) and the like.

Secretion

In some cases, it is desirable for the POI to be secreted from theexpression host into the culture medium from where the POI may be moreeasily recovered. According to the present invention, the secretionleader sequence may be selected on the basis of the desired expressionhost. Hybrid signal sequences may also be used with the context of thepresent invention.

Typical examples of heterologous secretion leader sequences are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g. from Aspergillus), the α-factor gene (yeastse.g. Saccharomyces and Kluyveromyces) or the α-amylase gene (Bacillus).

Detection

A variety of protocols for detecting and measuring the expression of thePOI are known in the art. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on thePOI may be used or a competitive binding assay may be employed. Theseand other assays are described, among other places, in Hampton R et al(1990, Serological Methods, A Laboratory Manual, APS Press, St PaulMinn.) and Maddox D E et al (1983, J Exp Med 15 8:121 1).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labelled hybridization or PCR probes fordetecting POI include oligolabelling, nick translation, end-labelling orPCR amplification using a labelled nucleotide. Alternatively, the NOI,or any portion of it, may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3 or SP6 and labelednucleotides.

A number of companies such as Pharmacia Biotech (Piscataway, N.J.),Promega (Madison, Wis.), and U.S. Biochemical Corp (Cleveland, Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. No. 3,817,837;U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No.3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 and U.S.Pat. No. 4,366,241. Also, recombinant immunoglobulins may be produced asshown in U.S. Pat. No. 4,816,567.

Additional methods to quantitate the expression of a POI includeradiolabeling (Melby P C et al 1993 J Immunol Methods 159:235–44) orbiotinylating (Duplaa C et al 1993 Anal Biochem 229–36) nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated. Quantitation ofmultiple samples may be speeded up by running the assay in an ELISAformat where the oligomer of interest is presented in various dilutionsand a spectrophotometric or calorimetric response gives rapidquantitation.

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the NOI is inserted within a marker genesequence, recombinant cells containing NOIs can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a NOI under the control of the promoter of thepresent invention or an alternative promoter (preferably the samepromoter of the present invention). Expression of the marker gene inresponse to induction or selection usually indicates expression of thePOI as well.

Alternatively, host cells which contain the NOI may be identified by avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridization and protein bioassay or immunoassay techniques whichinclude membrane-based, solution-based, or chip-based technologies forthe detection and/or quantification of the nucleic acid or protein.

SUMMARY

In summation, the present invention relates to a promoter and, also to aconstruct comprising the same. In particular the present inventionrelates to the use of a promoter for the expression of a NOI in anorganism.

In a preferred aspect, the present invention relates to the modificationof carbohydrate metabolism by the transformation (genetic manipulation)of a plant by use of the novel promoter of the present invention tocause expression of an NOI that affects carbohydrate metabolism.

In one preferred aspect, the present invention relates to themodification of galactomannan synthesis by the transformation (geneticmanipulation) of the leguminous plant Cyamopsis tetragonoloba (guar) byuse of the novel promoter of the present invention to cause expressionof an NOI that affects the galactomannan synthesis.

Deposits

The following sample was deposited in accordance with the BudapestTreaty at the recognised depositary The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive,Aberdeen, Scotland, United Kingdom, AB2 1RY on 15 Mar. 1999:

Microorganism Strain Number NCIMB Number E. coli TOP10 (Invitrogen) +NCIMB 41011 pTBR-ScaK3

NCIMB 41011 comprises the novel nucleotide sequence of the presentinvention.

A restriction map of the novel nucleotide sequence of the presentinvention is shown in FIG. 3.

The present invention also encompasses sequences derivable from thosedeposits and embodiments comprising the same. The present invention alsoencompasses partial sequences derivable from those deposits andembodiments comprising the same, wherein those partial sequences codefor regulatory elements.

The promoter of the present invention may be isolated from the depositby use of any of the suitable restriction enzymes indicated in FIG. 2.Alternatively, one could use PCR techniques to “PCR out” the promotersequence. Here, suitable primers would be based on the sequencepresented as SEQ ID No. 1.

By way of example, for amplification of the full-length RSus3 promoter(2700 bp) from pTBR-ScaK3, the primer-pair “M13 forward” and “RSusNcoI”can be used. The upper M13 forward primer anneals to the pCR2.1-TOPOpart of the clone some 110 bp from the cloning site, and the lowerRSusNcoI primer anneals to the translation start codon of RSus3.Alternatively the primer-pair “AP2” and “RSusNcoI” can be used toisolate a similar fragment (AP2 anneals to the adaptor part of thecloned fragment).

For amplification of the RSus3 promoter without intron 1 frompTBR-ScaK3, the primer-pair “AP2” and “Lowexon½” can be used. The lowerLowexon½ primer is designed as follows: The 3′ end of the primercorresponds to a 24 bp sequence just upstream of the 5′ splicing site inintron 1. The 5′ end of the primer corresponds to the 27 bp sequencebetween the 3′ splicing site and the ATG-codon, and incorporates of aNcoI site.

Hence, in summation, the present invention relates to a promoter that isuseful for causing selective expression of an NOI in the endospermtissue/cells of a transformed plant. The NOI and/or the promoter may beheterologous to the transformed plant—in the sense that the NOI and/orthe promoter may not naturally occur in the non-transformed plant. Thepromoter of the present invention is defined by having a nucleotidesequence corresponding to that shown as SEQ ID No. 1 or a variant,homologue, fragment or derivative thereof.

INTRODUCTION TO THE EXAMPLES SECTION AND THE FIGURES

The present invention will now be described, by way of example only,with reference to the accompanying drawings in which:—

FIG. 1 is a schematic diagram

FIG. 2 is a schematic diagram

FIG. 3 is a schematic diagram

FIG. 4 is a photograph

FIG. 5 is a schematic diagram

FIG. 6 is a schematic diagram

FIG. 7 is a schematic diagram

FIG. 8 is a schematic diagram

FIG. 9 is a schematic diagram

FIG. 10 is a schematic diagram

FIG. 11 is a graph

FIG. 12 is a schematic diagram

FIG. 13 is a schematic diagram

FIG. 14 is a schematic diagram

FIG. 15 is a photograph

FIG. 16 is a series of photographs

FIG. 17 is a graph

FIG. 18 is a graph

FIG. 19 is a schematic diagram

FIG. 20 is a schematic diagram

FIG. 21 is a schematic diagram

FIG. 22 is a schematic diagram

FIG. 23 is a schematic diagram

FIG. 24 is a schematic diagram

FIG. 25 is a schematic diagram

FIG. 26 is a graph.

Part I—Figures in More Detail

FIG. 1: A schematic representation (not to scale) of the promoter of thepresent invention and some of its associated sequences.

FIG. 2: Map of the RSus3 sequence in pScaK3 (pTBR-ScaK3) with thesurrounding restriction sites. The other clones are analogues to this,but some have the opposite orientation.

FIG. 3 is a restriction map of the promoter of the present invention(SEQ ID NO: 7).

FIG. 4: Agarose gel showing the result of the semi-nested PCR. Lane 1and 12 marker II; lane 2–6 EcoRV, Dral, PvuII, ScaI and SspI with bufferF: lane 7–11 same with buffer H.

FIG. 5. The promoter region from pScaK3 and pSspK3 were amplified fromthe primers M13 forward and RSusNco. The amplified products contain XhoIand NcoI sites for directional cloning into the unique SalI and NcoI inpGUSNOSt as XhoI/NcoI fragments.

FIG. 6 is a schematic diagram of a construct.

FIG. 7 is a schematic diagram of a construct.

FIG. 8 is a schematic diagram of a construct.

FIG. 9 is a schematic diagram of a construct.

FIG. 10 is a schematic diagram of a construct.

FIG. 11 shows a time course of sucrose synthase activity duringdevelopment of guar endosperm. The specific activity of sucrose synthasewere measured in extracts from guar endosperm at various developmentalstages from 7–41 days after flowering. The value of each point is themean sucrose synthase activities obtained from 3 to 4 independentmeasurements in which at least 5 endosperms from each pod were pooled.

FIG. 12: Presentation of sequencing plan for pTBR-ScaK3. The arrowsrepresent sequencing primers, and the numbers indicates approximateposition of 5′ end of the sequencing primers relative to the RSus3 ATGcodon.

FIG. 13: Schematic presentation of various RSus3/GUS/NOSt constructs.The numbers indicates the base pairs relative to the translational startcodon.

FIG. 14: Schematic presentation of various tandem Rsus3 constructs. Thenumbers indicates the base pairs relative to the translational startcodon.

FIG. 15: Agarose gel showing the result of control digest of tandemrepeat clones.

Lane 1: HindIII digest of pTandem leg/pro, which gives a specificfragment of 740 bp.

Lane 2: NheI digest of pTandem legumin, which gives a specific fragmentof 570 bp.

Lane 3: DNA marker VI. Lane 4: HindIII digest of pTandem prolamin, whichgis a specific fragment of 160 bp.

FIG. 16 a is a photograph of cotyledon tissue of guar that has beenballistically transformed with the ENOS control plasmid that containsthe GUS expression nucleotide sequence. This is a control study. As canbe seen, high levels of transient expression of GUS are observed.

FIG. 16 b is a photograph of endosperm tissue of guar that has beenballistically transformed with the ENOS control plasmid that containsthe GUS expression nucleotide sequence. This is a control study. As canbe seen, transient expression of GUS is observed.

FIG. 16 c is a photograph of cotyledon tissue of guar that has beenballistically transformed with the RSus3 construct plasmid p1730 (whichcontains a single copy of the nucleotide sequence of the presentinvention) that contains the GUS expression nucleotide sequence. As canbe seen, very low levels of transient expression of GUS are observed.

FIG. 16 d is a photograph of endosperm tissue of guar that has beenballistically transformed with the RSus3 construct plasmid p1730 (whichcontains a single copy of the nucleotide sequence of the presentinvention) that contains the GUS expression nucleotide sequence. As canbe seen, good levels of transient expression of GUS are observed.

FIG. 16 e is a photograph of cotyledon tissue of guar that has beenballistically transformed with the RSus3 construct plasmid pTandemleg/pro (which contains a tandem copy of the nucleotide sequence of thepresent invention) that contains the GUS expression nucleotide sequence.As can be seen, very low levels of transient expression of GUS areobserved.

FIG. 16 f is a photograph of endosperm tissue of guar that has beenballistically transformed with the RSus3 construct plasmid pTandemleg/pro (which contains a tandem copy of the nucleotide sequence of thepresent invention) that contains the GUS expression nucleotide sequence.As can be seen, high levels of transient expression of GUS are observed.

FIG. 17. Histogram showing the transient GUS expression in guarendosperms after bombardment with the RSus3 constructs. The data arepresented as mean number of blue spots per endosperm together with thecalculated standard error.

FIG. 18. Histogram showing the transient GUS expression in guar tissueafter correction for tissue size. The data are presented as mean numberof blue spots per cotyledon together with the calculated standard error.

Materials and Methods

Isolation of DNA

The plasmids used in this work were isolated using Qiaprep Spin MiniprepKit and Plasmid Maxi Kit from Qiagen (Hilden, Germany). These kits givevery clean plasmid preparations which are ready for use in cloning,sequencing, and transient expression assays. Further purification aretherefore not necessary. 10 mM Tris-Cl, pH 8.5 was used as elutionbuffer and storage buffer. All DNA was stored at −20° C.

The concentration of plasmid preparations was determined, by measuringthe absorbance at 260 nm in a Powerwave™ 200 spectrophotometer. An OD₂₆₀of 1 corresponds to about 50 μg/mL DNA. The amounts of DNA in isolatedfragments was estimated by comparative fluorescence of Ethidium-bromide(Etbr) stained DNA in agarose gels.

Restriction Enzymes

Enzyme digestion was performed using restriction enzymes from NEB andBoehringer M. The buffer provided by the supplier was used in thereactions. When double digestions were performed, a buffer compatiblewith both enzymes was chosen (e.g. digestions with both NheI and NcoIwas performed in NEBuffer 2 with BSA).

Electrophoresis

Electrophoresis was used to separate and identify DNA. The DNA wasstained with the fluorescent dye Ethidium bromide (EtBr) incorporated inthe gel in a concentration of 0.6 μg/mL. Appropriate amount of DNA wasmixed with loading buffer (0.1% bromophenol blue, 16% Ficoll 400) loadedon to the either Nusieve or Seakem agarose gels (FMC Bioproducts). TBEBuffer (0.1 M Tris, 0.09 M Boric acid, 0.001 M EDTA) was used aselecrophoresis buffer. DNA were separated by electrophoresis in a 0.5–2%(w/v) agarose gel, dependent upon the sizes of the bands of interest.For bands <1000 bp 2% Nusieve GTG agarose gels, and for bands >1000 bp0.5–1.5% Seakem LE agarose gels were used.

DNA molecular weight marker I, II, IV, VI and VII (Boehringer M) wereused for evaluation of electrophoreses DNA. The markers were provided ata concentration of 0.25 μg/μL and diluted as follows: 2 μL DNA marker, 2μL loading buffer and 8 μL H₂O. Either 4 μL or 10 μL of this mixture wasloaded on to gels depending upon the well size.

Selected bands were isolated from the gel, by minimal UV radiation,using Qiaquick Gel Extraction (Qiagen).

Ligation

All ligations were performed using T4 DNA ligase in the reaction buffer(66 mM Tris-HCl, pH 7.6, 6.6 MgCl₂, 10 mM dithiothreitol, 66 μM ATP)supplied by the manufacturer (Amersham Life Science). Fragment withCohesive ends were ligated using 1 U T4 ligase, whereas blunt-endedfragment were ligated using 5 U of the enzyme.

To prevent intramolecular religation of open plasmid, it was treatedwith alkaline phosphatase from the psychrophilic shrimp Pandalusborealis (Boehringer M). This enzyme catalyses the dephosphorylation of5′ phosphates from the plasmid DNA, but is easily prevented frominterfering with the subsequent ligation by heating to 60° C. for 15minutes.

Transformation

All the plasmids were used to transform, and propagated in,supercompetent TOP10 One Shot™ Cells (Invitrogen) Genotype: F mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 deoR recA1 araD139Δ(ara-leu)7697 galU ga/k rpsL endA1 nupG.

50 μL of supercompetent cells were carefully mixed with 1–5 μL ofligation mixture, and incubated on ice for 30 minutes, after which a 30second heat shock was performed, followed by a 2 minute recovery periodon ice. Finally, 250 μL of SOC medium were added, and the cells wereincubated in an orbital shaker at 37C for 30 minutes, before spreadingon to LB plates containing 100 μg/ml ampicillin.

Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a technique which is based on invitro amplification of a specific DNA sequence using synthetic primersflanking the sequence, and a thermostable DNA polymerase (e.g. Taq DNAPolymerase) for replication of the DNA.

A wide range of PCR techniques were extensively used in this study, e.g.Hot Start, Touch Down, Long PCR, Nested PCR and Sequencing PCR.

For optimization of PCR reactions the PCR optimizer™ Kit (Invitrogen)was applied. This kit comprises 16 buffers of four different pH values,each supplied with a range of four different magnesium ionconcentrations, and eases optimization of PCR reactions.

The following polymerases were used:

-   -   AmpliTaq GoId™ (Perkin-Elmer)    -   Expand™ High Fidelity PCR system (Boehringer M)    -   Taq DNA polymerase (Pharmacia Biotech)

Expand™ High Fidelity PCR system is a polymerase mix of the Taq and Pwopolymerases, and is designed for amplification of PCR products up to 12kb with high specificity. Taq posses 5′→3′ transferase activity andgenerates ends with single 3′ A overhang, whereas Pwo posses 3′→5′exonuclease activity and generate products with blunt ends.

With the exception of cycle sequencing, all PCR reactions reported hereused the Hot Start technique, which minimizes binding of primers topoorly matched sites on the template at low temperatures, and thesubsequent amplification of the spurious extension products formed inthe first cycle of the reaction. This is achieved by preventingpolymerisation before a substantial period of denaturation of thetemplate has elapsed, and this was effected in two different waysdepending on the type of polymerase.

When the Expand™ or AmpliTaq Gold™ DNA polymerase were used, a waxpellet was placed over the reaction mixture containing all componentsexcept for polymerase. The wax was melted by 95° C. for 2 min, followedby cooling to 4° C., which results in its hardening to form a thinsurface on top of the reaction mixture. On to this surface 1 μL of thepolymerase was added and the tube was then placed in the thermocycler,which was preheated to 95° C. beforehand, and the PCR programme wasstarted immediately.

AmpliTaq Gold™ is a thermally activated polymerase mixture which allowsa hot start, without the inconvenience of overlaying polymerase on to asolid wax surface. The enzyme is provided with an inactivating antibodybound to it. The polymerase is activated by a preliminary heating step,whereby the antibodies denatures, thus rendering the enzyme active. Thisallows a hot start PCR without the inconvenience of overlayingpolymerase on to a solid wax surface, and the denaturation time issignificant longer, and therefore more thorough. In these reactions allthe components were added together with wax pellet, and the reactionstarted as described above.

When amplification of long PCR products was desired, a time incrementwas added to the extension step in the PCR program.

The PCR reactions were performed in a Mastercycler (Eppendorf).

A typical example of such a reaction is summarised in the table below.

PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) Time*Template (1:100) 1 1 94  2′ 5*Buffer 10 2 94 30″ dNTP mix 4 3 68 30″Upper primer 4 μM 5 4 go to 2, 29 times Lower primer 4 μM 5 5 68 10″Water to 50 6 4 infinite Expand 0.75 *seconds: ″ and minutes: ′Cloning of PCR Products

Under most circumstances, isolated PCR products were cloned inpCR2.1-TOPO (Invitrogen) which was provided as a linearized fragmentwith single 3′-T overhangs and TOPO isomerase. This system exploits thetendency of Taq polymerase to add a single overhanging 3′-A in both endsof the amplified product The TOPO isomerase has both cleavage andligation activity, it cleaves the vector at a CCTTT site therebygenerating a single 3′-T overhang, which will religate with a PCRproduct with single 5′-A overhangs. This system therefore allows cloningof Taq amplified PCR products without the use of ligation with T4ligase.

The cloning site of pCR2.1-TOPO resides in a disruptive locus of thelacZ gene, which allows clones with inserts to be selected by blue/whitescreening in the presence of the chromogenic substrate forβ-galactosidase (x-gal). Miniprep DNA from white colonies was analysedby restriction digests, PCR and/or sequencing.

Some PCR-fragments were digested with enzymes and cloned directly intoplasmid vectors without a sub-cloning step. When cohesive ends could begenerated by restriction enzymes in this way, and exploited for directcloning, substantial savings in time were achieved.

Primers

The relevant primers are shown in the Tables presented below.

Primers and oligos (SEQ ID NOS 8–27, respectively, in order ofappearance) Listed in alphabetic order (Storage name, size) AP1:(Walking AP1a, 27-mer) 5′- GGA TCC TAA TAC GAC TCA CTA TAG GGC AP2:(Walking AP2, 17-mer) 5′- AAT AGG GCT CGA GCG GC Dellow1: (Dellow 1,22-mer) 5′ GCT TTC CAC CAC AAA ATG ACA C E356: (E356, 26-mer) 5′-GGA ATT CTA GTA ACA TAG ATG ACA CC E357: (E357, 28-mer) 5′- GGA ATT CCCCGA TCG TTC AAA CAT TTG G HindGCN4: (HindGCN4, 24-mer) 5′ GGA AGC TTGCGA AAA TGT GCA GGG Lowexon½: (Low exon ½, 56-mer) 5′ CCC ATG GCT ATCTTC TAG TTG GAT CCT CAA GCC TTG CAC TGA AGG GGA AGA GGA GG M13 forward:(M13 forward, 16-mer, TOPO Cloning Kit (Invitrogen)) 5′ CAT TTT GCT GCCGGT C M13 reverse: (M13 reverse, 17-mer, TOPO Cloning Kit (Invitrogen))5′ CAG GAA ACA GCT ATG AC Nhedel1: (Nhedel 1, 23-mer) 5′ CCG CTA GCA CAGAGG CTG AGC AG Nhedel2: (Nhedel.2, 27-mer) 5′ TGC TAG CTG GTA AAT GACATG CTG CTG Nhedel3: (Nhedel.3, 22-mer) 5′ CGC TAG CAG AGG CAG CAA GCT CNheGCN4: (Nhe GCN4, 22-mer) 5′ GGG CTA GCG AAA ATG TGG AGG G Oligo 1:(Walking Adaptor, 44-mer): 5′ CTA ATA CGA CTC ACT ATA GGG CTC GAG CGGCCG CCC GGG CAG GT Oligo 2: (Walking AP1b, 8-mer) 5′phosphate- AC CTGCCC - 3′amine pGUS lower: (pGUS lower, 22-mer) 5′ CTG GCG AAA GGG GGATGT GCT G Rsus3: (Lower RSus 3 Spe, 25-mer) 5′ ACG ACG GAA TGG ATA ATAGCA GAT A RsusNco: (RsusNco1, 21-mer) 5′ GTT TCC CCC ATG GCT ATC TTCRsusTATA: (RSusTATA, 21-mer) 5′ CCT CCC TGA AGC TTT TCG TGT UppCR2.1:(UppCR2.1, 44-mer) 5′ ATT AGG CAC CCC AGG CTT TAC ACT TTA TGC TTC CGGCTC GTA TG

Lower sequencing primers (SEQ ID NOS 28–34, in order of appearance)(Listed in numerical order): RSusseq 1 5′ Cy5- GT TTC CCC CAT TGC TATCTT C RSusseq 2 5′ Cy5- AG TGC CAG GTT CAA GGA CA RSusseq 700 5′ Cy5- ACCAA TCC CAG AAA CCC AAG C RSusseq 1000 5′ Cy5- GT GTC CCC TGC CTC ACT CCRSusseq 3 5′ Cy5- CC GGC TAA GTT AAA AAA AAA RSusseq 4 5′ Cy5- CT GTGCCG TTG GAA GCG TCA T RSusseq 5 5′ Cy5- CG CAG ATG GGT TCA GCC TTC A

Upper sequencing primers (SEQ ID NOS 35–39, respectively, in order ofappearance) (Listed in numerical order) Scaseq 1 5′ Cy5- GG TCG GCA CATTGA GAG GTC Scaseq 2 5′ Cy5- CA CAC CCA ACG CTC ACC GAT G Scaseq 3 5′Cy5- AG GAC GGT TTT GGT TGG GAT T Scaseq 4 5′ Cy5- TC CTC CTC TTC CCCCTT CAG TG Scaseq 5 5′ Cy5- AT CTG GCA ACC TTT TGT TTC T

M13 sequencing primers (SEQ ID NOS 40 and 41, respectively, in order ofappearance: M13 Reverse 5′ Cy5- CA GGA AAC AGC TAT GAC M13 Universal 5′Cy5- CG ACG TTG TAA AAC GAC GGC CAG TSequencing

The Rsus 3 promoter region was sequenced with a ALFexpress DNAsequencer. The ALFexpress is designed for automated detection offluorescently labelled DNA molecules separated by electrophoresis.

Cy5-labelled fluorescent M13 reverse and forward primers for thepCR2.1-TOPO vector was used for sequencing of 5′ and 3′ ends of clonedPCR products. In addition Cy5-labelled sequencing primers was designedfor specific regions of the Rsus3 promoter region, using the describedOLIGO™ program—Version 5.0 for Windows (National BioSciences Inc,Plymouth, Ma).

Materials for Sequencing

-   -   ALFexpreSST™ DNA Sequencer (Pharmacia biotech AB)    -   Thermo Sequenase fluorescent labelled primer cycle sequencing        kit with 7-deaza-dGTP (Amersham Pharmacia biotech)    -   ReproGel™ Long Read, for polyacrylamide gel electrophoresis with        the ALF® family of instruments (Amarsham Pharmacia Biotech)    -   ReproSet™, for UV polymerisation of Reprogel™ (Amersham        Pharmacia Biotech)    -   ALFwin™ Sequence Analyser 2.00, Windows 95 based program that        controls ALFexpress (Amersham Pharmacia Biotech)

Diodeoxy chain terminator sequencing was performed in a thermocycledreaction using the special mutant polymerase ThermoSequence supplied byAmersham Pharmacia Biotech which has been selected both to acceptfluorescently labelled primers and to have equal affinity for the fourdiodeoxy chain terminators. Band compressions on the gel were limited bythe use of 7-deaza-dGTP in the reaction.

Sequence Analysis

The resulting sequences was assembled using Winseq 1.01, developed by F.G. Hansen, Department of Microbiology, DTU, Denmark. After thispreliminary sequence assembling, the sequence analysis was performedusing the same program.

Isolation of the RSus 3 Promoter Region from Rice Genomic DNA

The rice sucrose synthase 3 (RSus3) promoter region was isolated fromrice genomic DNA using the chromosome walking technique described bySiebert et al. 1995, which is an adaptor mediated PCR method, designedto amplify an unknown sequence which is flanking a known sequence.

Genomic DNA is first digested with enzymes which produce blunt endedproducts (DraI, EcoRV, PvuII, ScaI or SspI) on to which adaptors areligated. The result is the generation of 5 DNA-libraries, which canserve as templates for a PCR, using one primer specific to the knownsequence and an adaptor-specific primer which is described in Siebert etal (ibid).

The adaptor sequence and the adaptor primer (AP1) and the nested adaptorprimer (AP2) was provided. The adaptor was designed with an aminogroupin 3′ end, which prevents amplification from the AP1 primer bindingsite, unless there is an initial round of amplification from e.g. agene-specific primer [After Siebert et al., 1995].

The adaptor incorporates two features which ensure that template withadaptor binding sites at both ends will not be amplified. Firstly, dueto an amino group in the 3′ end of the adaptor, the generation of an AP1primer binding site by extension of the lower strand 3′ end isprevented. Thus, the only circumstances in which an AP1 primer bindingsite is formed during the PCR, are those in which there is an initialround of amplification from a primer which binds within the restrictionfragment. Secondly, amplification of template, formed by unspecificpriming from AP1, will be suppressed. Fragments formed in this mannercontain inverted terminal repeats in the single stranded product, whichwill form a secondary stem-loop structure, which is more stable than thetemplate-primer hybrid. The formation of the stem-loop structureprevents annealing of primer, which suppresses amplification ofunspecific PCR products. The specificity of the method is furtherimproved by reamplification of the product using a nested PCR primer.

Preparation of Genomic DNA

Genomic DNA was obtained from rice. 1 μg of this DNA was digested with20 U of either of the following blunt end enzymes: DraI, EcoRV, PvuII,ScaI and SspI in a total volume of 50 μL.

The digestions were run through an enzyme remover column (Amicon) andconcentrated by alcohol precipitation followed by dissolving in 20 μLTE.

The adaptor was prepared by mixing 800 pmol of each of Oligo 1 and Oligo2 in a total volume of 22 μL. The mix was denatured by treatment at 94°C. for 1 min., and transferred on to ice.

An excess of the adaptor was ligated with the blunt-end digests using T4DNA ligase. 4 μL adaptor and 13 μL digest were mixed with 10 U T4 DNAligase in a total volume of 30 μL, and incubated at room temperature for24 hours.

Excess adaptors were removed after ligation with the Qiaquick PCRpurification kit (Qiagen), which recovers >100 bp PCR products andthereby also will remove excess adaptor.

PCR Screening for the RSus3 Promoter Region

For isolation of the RSus3 promoter region, the followingsequence-specific primer was designed:

-   5′-ACG ACG GAA TGG ATA ATA GCA GAT A-3′ (SEQ ID NO: 43)

The 3′ end of this antisense primer anneals approx. 300 bp downstream ofthe ATG of RSus3.

The amplification was performed in a Mastercycler (Eppendorf) as a hotstarted, two step, PCR (68°/94° C.). To ensure optimal product lengthand limitation of errors the proof-reading thermostable DNA polymerasemixture Expand™ (Boehringer M) was used.

For additional optimisation of the amplification, a PCR with one nestedprimer was performed. After the initial PCR (primer-pair AP1/Rsus3), asecondary semi-nested amplification (primer-pair AP2/Rsus3) wasperformed, using the product from the initial amplification as template.

The composition and reaction conditions for the initial PCR aresummarised in the table below.

Initial PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) Time*Template (1:20) 5 1 94 1′ 45″ 5x Buffer (E, F, G 10 2 94  15″ and H)dNTP mix (10 mM) 4 3 68  4″ AP1 (4 μM) 5 4 go to step 2, 9 times  Rsus3(4 μM) 5 5 94 115″ Water 20 6 68  4′ + 20″/cycle Expand ™ 0.75 7 go tostep 5, 19 times 8  4 infinite *minutes:′ and seconds: ″

Buffer E, F, G, and H for the PCR optimizer™ Kit (Invitrogen) werechosen for optimisation of the buffer conditions.

The semi-nested PCR was performed as Outlined in the table below.

Secondary PCR reaction PCR program Reagent μL/reac. Step Temp (° C.)Time* Template (1:100) 1 1 94 1′ 45″ 5x Buffer (F and H) 10 2 94 15″dNTP mix (10 4 3 68  4′ mM) AP2 (4 μM) 5 4 go to step 2, 9 times  Rsus3(4 μM) 5 5 94 15″ Water 25 6 68  4′ + 20″/cycle Expand ™ 0.5 7 go tostep 5, 19 times 8  4 infinite *minutes:′ and seconds: ″compatible with SalI and, since no internal XhoI sites are present inthe RSus3, this XhoI site represents the 5′ end of all of the nativeRSus3 fragments.

Sequencing revealed that the sequence around the ATG start codon inRSus3 was CAATGG. So a introduction of the NcoI (CCATGG) in theconsensus sequence around the ATG only affects a single basepair. Forgeneration of the 3′ end of the promoter region, the RSus3Nco primer wastherefore designed with a NcoI site positioned at the ATG start codonfor translational fusion of amplified promoter fragments with the uidAgene in pGUSNOSt.

For amplification of the two RSus3 fragments (1450 bp and 2700 bp) theprimer-pair M13 forward/RSusNco was used and the RSus3 fragments wereamplified from pSspK3 and pScaK3. The M13 primer anneals to thepCR2.1-TOPO part of the pSsp and pSca clones some 110 bp from thecloning site.

The two RSus3 fragments were amplified according to the followingscheme:

PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) TimeTemplate* (1:20) 5 1 95 2′ 5x Buffer (G and H) 10 2 95 1′ dNTP mix (10mM) 4 3 55 1′ M13 forward (4 μM) 5 4 72 2′ RsusNcol (4 μM) 5 5 go to 2,25 times Water 20 6 72 10′  Expand ™ 0.5  4 0′ *Qiagen miniprep ofpSspK3 or pScaK3

The resulting products were cloned in pCR2.1-TOPO and then subclonedinto pGUSNOSt as XhoI/NcoI fragments. Positive clones were identified byrestriction mapping of miniprep DNA. Although the pGUSTNOSt waslinearised with both SalI and NcoI, the vector was treated withphosphate before ligation to prevent religation of plasmid that was cutwith only one enzyme due to the close placement of the SalI and NcoIsites (G/TCGAC/CATGG) (SEQ ID NO: 43).

The NOS terminator was amplified from pDB2 as a 266 bp fragment with theE357/E356 primerpair, each of which contained an EcoRI site 5′ to theannealing region. The amplified fragment was cloned in pCR2.1-TOPO, apositive clone was identified, and named pNOSt9. This was sequenced inboth directions with universal and reverse sequencing primers(pCR2.1-TOPO) and the resulting sequence was verified using the BLASTfunction in the Entrez search engine. NOSt was excised from pCR2.1-TOPOwith EcoRI and cloned into the unique site for this enzyme inpGUSN358→S. The orientation of the fragment in pGUSN358→S was tested byPCR using the primer pairs E357/pGUSlower for the correct orientation,and E356/pGUSlower for reverse orientation (product of 350 bp). A clonewith NOSt in the correct orientation was selected, and named pGUSNOSt 2(5253 bp), which was later just referred to as pGUSNOSt, whereas a clonewith NOSt in reverse orientation was selected and named pGUSNOSt1.

Cloning of the RSus3 Promoter in pGUSNOSt

The initial goal was to clone as much as possible of RSus3 promoter intopGUSNOSt, without any change of the promoter sequence.

Restriction mapping of the RSus3 promoter localised the followingfeatures:

-   1. An internal HindIII site approx. 1200 bp upstream the ATG codon.-   2. A PstI site in clones originating from rice DNA template digested    with Sca I (pSca clones), but not in those digested with Ssp I (pSsp    clones).-   3. Several SphI sites in both pSca and pSsp clones.-   4. No SalI and NcoI sites were found in any of the cloned fragments.

These characteristics limited the number of unique restriction sites forcloning of the RSus3 promoter in pGUSNOSt. Only SalI and NcoI wereavailable for cloning of the RSus3 promoter.

The cloning strategy was to generate two RSus3 upstream fragments (1450bp and 2700 bp) with ends compatible to the SalI and NcoI sites fordirectional and translational fusion of the RSus3 promoter with the uidAgene. The cloning strategy is outlined in FIG. 5.

The adaptor from the chromosome walking technique contains a XhoI siteand therefore the isolated RSus3 promoters in the pSsp and pSca clonesall contain this XhoI site in the 5′ end of the promoter region.Overhanging ends generated from cutting with XhoI are compatible withSalI and, since no internal XhoI sites are present in the RSus3, thisXhoI site represents the 5′ end of all of the native RSus3 fragments.

Sequencing revealed that the sequence around the ATG start codon inRSus3 was CAATGG. So a introduction of the NcoI (CCATGG) in theconsensus sequence around the ATG only affects a single basepair. Forgeneration of the 3′ end of the promoter region, the RSus3Nco primer wastherefore designed with a NcoI site positioned at the ATG start codonfor translational fusion of amplified promoter fragments with the uidAgene in pGUSNOSt.

For amplification of the two RSus3 fragments (1450 bp and 2700 bp) theprimer-pair M13 forward/RSusNco was used and the RSus3 fragments wereamplified from pSspK3 and pScaK3. The M13 primer anneals to thepCR2.1-TOPO part of the pSsp and pSca clones some 110 bp from thecloning site.

The two RSus3 fragments were amplified according to the followingscheme:

PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) TimeTemplate* (1:20) 5 1 95 2′ 5x Buffer(G and H) 10 2 95 1′ dNTP mix (10mM) 4 3 55 1′ M13 forward (4 μM) 5 4 72 2′ RsusNcol (4 μM) 5 5 go to 2,25 times Water 20 6 72 10′  Expand ™ 0.5  4 0′ *Qiagen miniprep ofpSspK3 or pScaK3

The resulting products were cloned in pCR2.1-TOPO and then subclonedinto pGUSNOSt as XhoI/NcoI fragments. Positive clones were identified byrestriction mapping of miniprep DNA. Although the pGUSTNOSt waslinearised with both SalI and NcoI, the vector was treated withphosphate before ligation to prevent religation of plasmid that was cutwith only one enzyme due to the close placement of the SalI and NcoIsites (G/TCGAC/CATGG) (SEQ ID NO: 43).

A positive 1450 bp clone was named “p1450” and a positive 2700 bp clonewas named “p2700”.

Deletions of the RSus3 Promoter

In order to identify those parts of the cloned promoter regions whichconfer activity and specificity, we performed a molecular dissection ofthe cloned fragments, by effecting serial deletions of regions whichwere deemed from the DNA sequence to contain motifs, identified in thepromoters of other genes.

The generation of RSus3 promoter deletions involved PCR amplification ofselected parts of the promoter region, and cloning of these fragmentsinto a construction which was based on the p1450 construct. It wasdecided that all truncations of the promoter would be upstream of theTATA box, which is positioned 989 bp 5′ to the translation start codon,and approximately 100 bp upstream of intron 1. This strategy gave theoption of retaining or later removing intron 1 from constructs, whilefocussing deletions upon the region in which all the recognised promotermotifs are located.

As described above, there are a limited number of restriction sitesavailable in the pGUSNOSt MCS for cloning of truncated promoter inserts.In fact, there are no unique sites left in the pGUSNOSt part of theresulting clones. But in the adaptor part of the cloned 1450 bp and 2700bp fragments there are a number of restriction sites.

Only a few restriction sites are unique within the RSus3 part of thep2700 and the p1450 constructs. Of these, only BamHI, BglII, BsiWI andClaI generate cohesive ends and are therefore convenient for cloning,but BamHI is located downstream of intron 1 and BglII within it. TheBsiWI and ClaI on the other hand are located only 300–400 bp from theMCS and therefore leave very little sequence that can be truncated.

So the lack of usable cloning sites led to the following strategy:

The MCS HindIII site was converted to a NheI site by partial digestionfollowed by fill-in with Klenow and religation of the resulting bluntended fragment. PCR amplified fragments with NheI/HindIII couldafterwards be cloned directionally in the resulting unique NheI andHindIII sites.

This strategy had several advantages, though it is quite laborious.

-   1) A standard test plasmid was created, which contained unique NheI    and HindIII sites, into which PCR amplified products could be    directionally cloned.-   2) The sequence of the HindIII/NcoI fragment obtainable from all    truncated clones was identical, so possible variations owing to    PCR-generated mutations in this region were avoided.    HindIII→NheI Conversion for Directional Cloning of PCR Deletions

The conversion of the HindIII site in the MCS to a NheI site involvedpartial digestion with HindIII, filling-in of the 5′ single strandedoverhang with Klenow fragment, and religation which formed an NheI site.Reaction conditions for effecting a partial digestion with Hind III wereestablished empirically, using the range of dilutions of enzyme in thetabel below and slowing the reaction by performing it at 25° C., ratherthan 37° C.

Reaction scheme for the partial digestion: HindIII Plasmid NEBuffer 2dilution¹ H₂O sample dilution¹ (μL) (μL) (μL) (μL) 1 2 1,5 2 9,5 2 2 1,54 7,5 3 2 1,5 6 5,5 4 2 1,5 8 3,5 5 2 1,5 10 1,5 6 2 1,5 12 0 Dilutions:0.25 μg/μL plasmid and 0.02 U/μL HindIII

The partial digests were incubated in a Mastercycler (Eppendorf) for 15min at 25° C. followed by 10 min at 65° C. The result was evaluated on a1% Seakem agarose gel and sample 2 and 3 gave the best result. In thesetwo samples there were 4 bands: 2 bands from uncut plasmid, a band froma single HindIII cut and a faint band from cutting of both HindIIIsites. The latter band increased in intensity in the subsequent samples,at the expense of the single cut band.

The partially HindIII-digested 1450 bp construct was filled in using DNAPolymerase I Large Fragment (Klenow) (NEB) before ligation. In thepresence of dNTP's, Klenow fragment retains the polymerization fidelityof the E. coli DNA Polymerase I without degrading 5′ termini. Treatmentwith Klenow fragment was performed as follows:

-   -   14 μL partial digested plasmid    -   2 μL 10 mM dNTP mix (Invitrogen)    -   0.6 μL NEBuffer 2 (NEB)    -   2.9 μL H₂O    -   0.5 μL Klenow fragment (NEB)

The reaction was incubated for 15 min at 25° C. and 10 min at 75° C.After the filling-in reaction the singly cut plasmid band was isolatedfrom the gel, ligated and positive clones were isolated. This strategygives rise to two different types of clones, one with an upstream NheIsite (p2700(upNheI) 1 and 3), and one with a downstream NheI site(p2700(downNheI) 7 and 10). Although the latter type of clone was notsuitable for promoter truncation studies, it found utility later duringthe construction of tandem repeats of promoter motifs.

Additionally, the HindIII site in the MCS of the parent plasmidpGUSNOSt, which contained no RSus3 sequence, was modified to an NheIsite in a similar manner, to form pGUSNOStNheI 2 and 3, but the partialdigestion was of course not necessary for the unique HindIII site inthis plasmid.

Cloning of Three RSus3 Deletions in p2700(upNheI)

See FIG. 6.

Three deletions were generated using the three primer-pairs withdifferent upper primers, but the same lower primer: Nhedel1/Dellow1,Nhedel2/Dellow1 and Nhedel3/Dellow1. The Nhedel1–3 primers was designedwith a NheI site and the Dellow1 primer was designed to annealdownstream of the internal HindIII site. Thus these primer-pairs resultsin generation of 3 different PCR products (650 bp, 590 bp and 480 bp),with the 2700 bp construct as template, each with NheI/HindIII sites forcloning in p2700(upNheI).

The amplification was performed in a Mastercycler (Eppendorf) as a2-step PCR (68/94° C.) described in the table below.

PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) Time Template(1:100) 1 1 95  2′ 5x Buffer (E, F, G or 10 2 94 45″ H) dNTP mix (10 4 368 45″ mM) Nhedel1, 2, 3 (4 5 4 go to 2, 25 times μM) Dellow1 (4 μM) 5 568 10′ Water 24,5 6  4 infinite Expand ™ 0.5

The PCR products were cloned in pCR2.1-TOPO and positive clones wereidentified by restriction mapping. NheI/HindIII fragments from thesewere isolated and cloned into p2700(upNheI) 1.

Three positive clones were identified and named “p1730” clone 3, “p1670”clone 3 and “p1560” clone 3.

The p1160 Construct

See FIG. 7.

This deletion was generated by cutting the p2700 construct HindIII. Thevector was religated and two positive clones were named “p1160” clone 1and 2.

The p2700-Prolamin Construct

See FIG. 8.

Whereas all the RSus3 deletions described above are 5′ end deletions,this truncation comprises an internal deletion of 161 bp between theinternal HindIII site and the TATA-box in the RSus3 promoter. Based onthe RSus3 sequence, a primerpair (RsusTATA/RsusNco) was designed foramplification of a 1020 bp fragment, with a HindIII site at one end andan NcoI site at the other. This product therefore comprises Intron 1flanked by the TATA box, and the ATG translational start codon. Theprimer RsusTATA contained an internal HindIII site and annealed justupstream the TATA-box. The primer RsusNco, which contains a NcoI sitepositioned at the ATG start codon for translational fusion of amplifiedpromoter fragments with the uidA gene, was the same as described abovein section 5.

The PCR reaction was performed as a 2-step PCR (68/94° C.) after thefollowing scheme in a Mastercycler (Eppendorf).

PCR reaction PCR program Reagent μL/reac. Step Temp (° C.) Time Template(1:100) 1 1 95  2′ 5x Buffer (A and B) 10 2 94 45″ dNTP mix (10 mM) 4 368 45″ RsusTATA (4 μM) 5 4 go to 2, 30 times RsusNco (4 μM) 5 5 68 10′Water 24,5 6  4 infinite Expand ™ 0.5

The 1020 bp HindIII/NcoI product was generated by PCR and cloned intopCR2.1-TOPO. Positive clones was identified, and the HindIII/NcoIfragment was cloned into 2700 bp (upNheI) (linearized with HindIII andNcoI for substitution of a corresponding 1160 bp fragment). A positiveclone was isolated and named “p2700-prolamin” clone 1.

Removal of Intron 1 within the RSus3 Promoter Region

In order to create a promoter lacking Intron1, screening for the5′-untranslated region of an RSus3 clone in a rice cDNA library, andsplicing of this to the upstream promoter motifs already cloned, wereinitially considered. However, sequence analysis revealed the 3′acceptor splice site of intron 1 to be located only 27 bp upstream ofthe ATG-codon. In light of this, a preferred strategy was formulated inwhich the sequence of this 27 bp downstream non-coding exon wasincorporated in the lower strand PCR primer used to amplify thepromoter.

See FIG. 9.

A primer pair was designed for PCR amplification of the RSus3 promoterregion from pSca and pSsp clones. The lower primer (Lowexon½) wasdesigned as follows: The 3′ end of the primer corresponded to a 24 bpsequence just upstream for the putative 5′ splicing site in intron 1.The 5′ end of the primer corresponded to the 27 bp sequence between the3′ splicing site and the ATG-codon (NcoI). The upper primer UppCR2.1 wasdesigned to anneal to a 44 bp sequence of pCR2.1-TOPO, upstream of theM13 reverse primer site, for amplification of the RSus3 promoter withoutintron 1 from pSca and pSsp clones.

Both primers were quite long (Lowexon½ is 56 bp and UppCR2.1 is 44 bp),and to ensure that they were full length, they were ordered HPLCpurified. Lowexon½ has the possibility to form a hairpin loop with amelting temperature of 56° C. and an internal BamHI site gives a stableprimer dimer, so the PCR amplification was performed using AmpliTaq Goldan a two step program (68° C./94° C.).

Due to the potential of these long primers to form partiallymiss-matched double-stranded secondary structures at their 3′ end,AmpliTaq gold was used instead of Expand™ polymerase, in order to avoidthe modification of the oligonucleotides by the proof-reading Pwopolymerase.

The result was a very faint band of the right size, so a semi-nested PCRamplification with M13 reverse/RsusNcoI was performed using purifiedproduct as template.

This nested PCR gave a more intense band, which was cloned inpCR2.1-TOPO. A positive clone was identified and a XhoI/NcoI fragmentwas subcloned in pGUSNOSt (Analogous to subcloning of XhoI/NcoIfragments from pScaK3 and pSsp3). A positive clone was named“p1450-intron” clone 2.

Generation of Tandem-Repeat RSus3 Promoters

In an attempt to enhance the activity of the RSus3 promoter, a range ofsequences upstream the TATA-box in RSus3 were doubled. This strategyresulted in 3 tandem-repeat RSus3 promoters, in which different parts ofthe RSus3 promoter region had been doubled.

See FIG. 10.

DNA sequence analysis located the majority of putative cis-elements tobe within 700 bp (−1709 bp to −1027 bp), upstream of the TATA box.Therefore these 700 bp contained the elements to be doubled.

The three tandem-repeat promoters were constructed by amplification ofspecific sequences of the promoter, cloning of these into pCR2.1-TOPO,and finally subcloning the cloned fragments in either the NheI site orthe HindIII site in the p1730 construct.

Tandem Legumin

The repeated sequence in the tandem legumin promoter were amplifiedafter same description as NheI/HindIII product in p1730, but withp2700(downNheI) as template. This gave a similar product, only with twoNheI sites, which after cloning into pCR2.1-TOPO, was subcloned as anNhe I fragment into the NheI site of p1730. A positive clone wasidentified by NheI digestion, and a the orientation was established byrepeating the PCR using the subclone as template. The resulting clonewas named “pTandem legumin.”

pTandem Leg/Pro

This tandem-repeat promoter was constructed in a similar approach. Aprimer pair was designed for amplification of a 750 bp fragment from thep2700 construct. The primer pair each had an NheI site (Nhedel1 used forp1730, and NheGCN4). Amplification of the primer pair Nhedel1/NheGCN4,was again performed, after same description as for the p1730 construct,and resulted in a 750 bp product (with NheI sites in both ends), whichwere cloned in pCR2.1-TOPO, and afterwards subcloned into the NheI siteof the p1730 construct. A positive clone was identified by restrictiondigest, the orientation was determined in the same manner, and the clonewas named “pTandem leg/pro”.

pTandem Prolamin

This tandem-repeat promoters was generated in almost the same manner asthe tandem leg/pro. The primerpair had same upper primer, and ananalogous lower primer with a HindIII[[site (HindGCN4). The resultingproduct was cloned into pCR2.1-TOPO and a 170 bp HindIII/HindIIIfragment from this was subcloned in the HindIII site in the p1730construct. A positive clone was identified by restriction digest, theorientation of which was determined by repeating the PCR using thesubclone as template The resulting clone was named “pTandem prolamin”.

Transient Ballistic Transformation with the Particle Inflow Gun (PIG)

The Particle Inflow Gun (PIG) used in this study was in accordance withFiner et al. 1992 and Vain et al. 1993. The PIG—which is placed on asterile-bench for minimizing contaminants—comprises a vacuum chamberwith a digital vacuum sensor, an electronically operated solenoid gasvalve attached to a helium cylinder regulator (1–16 bar), and athree-way valve connected to a vacuum pump. The solenoid regulates thehelium flow, and is controlled by a timer relay which allows gated flowin pulses down to 25 milliseconds. The PIG is operated from a controlpanel with a timer relay, a digital display vacuum gauge and afire-button.

The vacuum chamber is constructed from 3 mm stainless steel with a 20 mmacrylic door and a 10 mm inner lining of polypropylene with grooves atevery 20 mm for a shelf. A silicone foam gasket between the chamber andthe door seals the vacuum chamber.

A ballistic transformation device, similar to the PIG, was used in apreliminary experiment to test the feasibility of transformation of guarendosperms by particle bombardment.

Preparation of Guar Tissue for Transformation

The primary target for particle bombardment in this study was the guarendosperm, but both emerged and pre-emerged cotyledons, as well asjuvenile leaves, were also used as comparative tissue. Endosperms andemerged cotyledons were isolated from 3–4 weeks old guar pods,pre-emerged cotyledons were harvested from 12 and 20 day old in vitrogrown seedlings, and juvenile leaves were harvested from mature guarplants.

Isolation of Guar Tissue

Endosperms were isolated from guar seeds, under aseptic conditions,after the following procedure:

The sterilised guar pods were opened, and the seeds taken out. For eachseed, the coat was carefully removed, and the remaining part was dividedin endosperm and embryo. The endosperm was divided in two identicalhalves, and the convex dorsal side of both halves was the target surfacefor particle bombardment. This bisection and orientation minimiseddispersal of the tissue by the helium blast. The pre-emerged cotyledonswere separated from the embryo, whereas emerged cotyledons were isolatedfrom seedlings grown in vitro in a 12 hour day/night regime. Young guarleaves were excised from full-grown plants.

The isolated tissue was transferred to the 60 mm petri dishes containingsolid agarose medium and a filter paper disc (see section 1.4.3). Thetissue were placed as follows: Endosperms in the centre of the petridish with dorsal side up; cotyledons from seedlings with the upper sidefacing up in the centre of the petri dish; Embryo cotyledons togetherwith endosperms.

A particle bombardment experiment consisted of 5 to 6 repeatedbombardments per construct. 6–8 endosperms, 2–3 cotyledons fromseedlings, or 6–7 endosperms plus 4–5 pre-emerged cotyledons werebombarded in each discharge of the PIG. For comparative analysis, bothcotyledons and endosperms were bombarded in same experiment.

Sterilisation of Guar Tissue

No antibiotics were used in the transformation, so microbialcontamination must be avoided. Therefore a fastidious sterilisationprocedure was very important.

Sterilisation of guar pods from mature guar plants were accomplished asfollows: The pods were washed in sterile water, submerged for 1 min in96% EtOH, and finally washed 3 times with sterile water. Furthersterilisation of the guar pods and seeds was not necessary.

Sterilisation of new leaves from mature guar plants was accomplished asfollows: The leaves were washed in sterile water, submerged for 10s in96% EtOH and for 20 min in 0.15% NaOCl and 0.05% Triton X-100. Finallythe leaves were washed 3 times with sterile water, and stored in wateruntil use.

Cotyledons from in vitro grown seedling were sterile and nosterilisation was necessary.

Medium

The tissue was bombarded and incubated on the following medium. Themedium was a solid medium, and was based on a recipe from Donovan & Lee1977, with a few modifications in the concentration of the ingredients.

Concentration Compound (mg/L) Salts Sucrose 20,000 M&S mix¹ 4,300Vitamins Myo-inositol 100 Thiamine 0.4 Aminoacids L-Alanine 89L-Arginine 126.5 L-Asparagine 117 L-Aspartic acid 100 L-cysteine 69L-glutamic acid 664.5 L-Glutamine 665.5 Glycine 156 L-Histidine 112L-Isoleucine 174 L-Leucine 274.5 L-Lysine 77 L-Methionine 67L-Phenylalanine 287.5 L-Proline 443.5 L-Serine 202.5 L-Threonine 96L-Tryptophane 164 L-Tyrosine 144 L-Valine 161.5 Agar Agar 8,000 type A1296 ¹Murashige & Skoog basal salt mixture M5524The Particle Bombardment Procedure

The particle bombardment experiments in this study had been performed asdescribed below.

DNA Coating of Gold Particles

A typical transformation with a single plasmid, comprising five repeateddischarges of the PIG, employed 3 mg of gold particles. In practice, alarger batch of gold particles than this was freshly prepared at onetime, and used for several transformations immediately afterwards. Theprocedure described below for the preparation of 3 mg of gold particleswas adjusted such that amounts and volumes increased in proportion toamount of material.

-   1. 3 mg of gold particles (1.6 μm, Biorad) were treated with 50 μL    99.9% EtOH (Danisco Distillers).-   2. The gold particles were vortexed in the ethanol for 3 min (full    speed) and centrifuged at 10.000 g for 1 min.-   3. The supernatant was removed and resuspended in 50 μL of sterile    H₂O.-   4. The particles were centrifuged at 10.000 g for 1 min, the    supernatant was removed and this washing with water repeated.-   5. The particles were resuspend in 50 μL 50% w/v glycerol (sterile).

The gold particles were then coated with plasmid DNA in the followingmanner.

-   1. 50 μL of gold particle suspension were removed during vortexing    (14.000 min⁻¹) on an thermomixer (Eppendorff).-   2. While vortexing (14.000 min⁻¹) this 50 μL sample of gold    particles, the following components were sequentially added.    -   10 μL of plasmid (10 μg/μL)    -   50 μL 2.5 M CaCl₂    -   20 μL 0.1 M Spermidine-   3. Vortexing was continued for 3 min at 16.000 min⁻¹. The sample was    then sedimented at 10.000 g for 10 s. and the supernatant was    removed.-   4. The sample was resuspended in 250 μL 99.9% EtOH, and centrifuged    at the same force for 10s.-   5. The supernatant was removed and the particles resuspended in 40    μL 99.9% EtOH.-   6. The DNA coated microprojectiles were stored at ice until use.

Because the gold particles tend to agglomerate irreversible in aqueoussolutions, it was necessary to prepare them immediately prior to use[Kikkert 1993]. CaCl₂ can be store at 4° C., but spermidine(N-β-aminopropyl]-1,4-butanediamine) deaminates with time, and solutionsmust be freshly made every month and be stored frozen at −20° C.

The purpose of the spermidine in the coating procedure is to condensethe DNA by shielding the negative charges on the DNA phosphate backbone,thereby allowing hydrophobic interactions to predominate [Bloomfield1991].

Operating the PIG

The PIG was operated in accordance with the following procedure

-   1. A petri-dish (60 mm in diameter) with the target tissue was    placed in the vacuum chamber.-   2. A stainless steel mesh (250 μm-mesh, 13 mm in diameter) was    placed in the filter unit.-   3. 5 μL of a suspension of DNA-coated micro-projectiles were loaded    on to the centre of the steel mesh.-   4. The filter unit was assembled and attached to the helium solenoid    valve.-   5. The 250 μm-mesh stainless steel protection screen was placed on    top of the petri-dish.-   6. The door and the valves were closed and evacuation of the chamber    was begun.-   7. The helium burst was released by pressing the fire button exactly    at the point when the desired vacuum was reached.-   8. The vacuum was released by opening the evacuation port, and the    tissue was removed.-   9. The cycle was repeated until all the samples had been bombarded.    The filter unit, mesh and protection screen were changed for every    new construct.    Bombardment Conditions

Good results for ballistic transformation of guar endosperms, embryos,and cotyledons were obtained with the following bombardment conditions.

-   -   The vacuum chamber was evacuated to a partial vacuum of 0.1 bar.    -   The target was placed at a distance of 16 cm, measured from the        mesh in the filter unit to the target tissue.    -   The helium pressure on the regulator was set at 5 bar.    -   The timer relay was set at 50 milliseconds.    -   A stainless steel protective screen (250 μm-mesh) was placed        approx. 2 cm above the target tissue    -   Gold micro-projectiles with a diameter of 1.6 μm (Biorad) were        used to carry the plasmid DNA.        Post Bombardment

After bombardment the petri-dishes, containing bombarded tissue, weresealed and incubated for 48 hour at 25° C. Endosperms and embryos wereincubated in the dark, leaves and cotyledons were incubated in a 12 hourday/night regime.

GUS Assay

The histochemical GUS assay was performed as described by Jefferson1987. After the incubation period the bombarded tissues were transferredto microtitre plates, submerged in GUS-assay buffer and incubated indark for 24 hours at 37° C.

GUS-Assay Buffer:

-   -   100 mM Sodium-phosphate buffer, pH 7.5    -   0.5 mM Potassium ferricyanide (III)    -   0.5 mM Potassium ferrocyanide (II)    -   10 mM Na₂EDTA (Titriplex III)    -   1.9 mM X-Gluc

A method for determination of GUS expression, and thereby promoteractivity, by counting the number of blue spots was adapted from Knudsen& Müller [1991]. After bombardment, incubation and GUS-assay the numberof blue spots (expression units) was counted under a microscope, andexpressed relative to the area examined.

The cotyledons and leaves were cleared in 96% EtOH in several washingsteps, and were stored in 70% EtOH until counting of blue spots.

Blue spots in endosperms had to be counted immediately after the GUSincubation period. Neither storage in alcohol nor water were applicable,due to high content of galactomannan and water. The water causedswelling of the endosperm and ethanol made the endosperm shrink. Thesechanges destroyed the expression pattern on the surface of theendosperm.

Presence of Sucrose Synthase in Guar Endosperm

A typical developmental increase in sucrose synthase, that coincideswith seed-fill, was verified in extracts from guar endosperm using acontinuous sucrose synthase assay (see FIG. 11). Details on the sucrosesynthase assay are as follows.

Sucrose Synthase Assay

The sucrose synthase activity in developing guar endosperms was assayedin the direction of sucrose cleavage. The sucrose synthase catalyses thecleavage of sucrose in the presence of UDP into fructose andUDP-glucose:sucrose+UDP

fructose+UDP-glucose

The activity of sucrose synthase was assayed by monitoring the formationof UDP-glucose in a continuous enzyme reaction described by Keller etal. (Keller F, Frehner M & Wiemken A (1988) Sucrose Synthase, aCytosolic Enzyme in Protoplasts of Jerusalem Artichoke Tubers(Helianthus tuberosus L.) Plant Physiol. 88, 239–241). The formation ofUDP-glucose was coupled to the reduction of NAD⁺ in the presence ofUDP-glucose dehydrogenase (E.C. 1.1.1.22), which catalysis oxidation ofUDP-glucose to UDP-glucuronic acid:UDP-glucose+2 NAD⁺+H₂O

UDP-glucuronic acid+2 NADH

The endosperms were dissected from the developing seeds, pooled andhomogenised in Hepes/KOH buffer (20 mM, pH 8.00) using a rotatingpestle. During this procedure enzymatic breakdown of the galactomannanbackbone was effected by addition of β-mannase (E.C. 3.2.1.78,Megazyme). The sample was centrifuged at 20,000 g for 20 minutes and theresulting supernatant was desalted using Sephadex® G-25 columns (NAP™-5,Pharmacia Biotech), for removal of endogenous sugars from the crudeextract.

An appropriate amount of enzyme extract was added to the sucrosesynthase assay buffer (1 ml):

-   -   20 mM Hepes/KOH (pH 8.0)    -   200 mM sucrose    -   2 mM UDP    -   1.5 mM NAD⁺    -   20 mU UDP-glucose dehydrogenase (Sigma)

The reduction of NAD⁺ to NADH was followed by continuous measurement ofthe absorbance at 340 nm in a spectrophotometer (25° C., ε_(NAOH)=6300 lmol⁻¹ cm⁻¹). The amount of UDP-glucose produced (μmol min⁻¹) wascalculated from the slope of the resulting absorbance curve. The totalamount of protein, in the remaining supernatant, was determined usingthe Biorad Protein Assay with bovine serum albumin as standard, and thespecific activity of sucrose synthase was calculated (Units sucrosesynthase per mg protein, or μmol min⁻¹ mg⁻¹).

The activity of sucrose synthase was measured in extracts from guarendosperms at various developmental stages from 7–41 days afterflowering. The mean sucrose synthase activity for each developmentalstage was obtained from 3 to 4 independent measurements, each of whichwas made with extract obtained from at least 5 endosperms from same pod.

Results

Isolation of the RSus3 Promoter Region

The rice sucrose synthase 3 (RSus3) promoter region was isolated fromrice genomic DNA using the chromosome walking technique described bySiebert et al. 1995. After the semi-nested PCR reaction, 3 bandsappeared in the following reactions: DraI (≈700 bp), ScaI (=3000 bp) andSspI (≈1800 bp).

The 1800 bp and 3000 bp products were verified, as specific productsobtained from the primer-pair AP2/RSus3, by single primer controlreactions.

Template\Primer AP2 RSus3 AP2/RSus3 Scat (buffer H) — — ≈3000 bp Sspl(buffer H) — — ≈1800 bp

The result of this single primer control reaction shows that the 1800 bpand 3000 bp originated from specific amplification from the AP2/Rsus3primer-pair and are not single primer products. Single primer productfrom the AP2 is most likely the result of amplification from twoadaptors.

The 1800 bp and 3000 bp products were cloned into pCR2.1-TOPO. Nineclones of the 1800 bp ligation and seven clones of the 3000 bp ligationwere isolated. Restriction mapping with EcoRI showed that of these, four1800 bp clones and six 3000 bp clones had insert between the two EcoRIsites.

The resulting pCR2.1 clones with 1800 bp and 3000 bp inserts werepartially sequenced using M13 universal and reverse sequencing primersspecific for pCR2.1-TOPO. Downstream sequences for the 1800 bp and 3000bp clones were compared to the published part of the RSus3 sequence(Huang et al. 1996 (GenBank accession number: L03366)] using thealignment feature in the Winseq program.

All four 1800 bp clones were shown to have the upstream part oftranscribed region of RSus3 inserted (two clones of each orientation).These clones were named pSsp A2, K1, K3 and K4.

Four 3000 bp clones were also shown to have the upstream part of thetranscribed region of RSus3 inserted (with both orientationsrepresented). These clones were named pSca K3, K5, New1 and New5.

See FIG. 2.

Presentation of Sequence Data

The clone pTBR-ScaK3 (pScaK3) was chosen for total sequencing of theRSus3 promoter region. This clone was sequenced on both strands afterthe sequencing plan outlined in FIG. 12.

The other positive RSus3 clones described above was partially sequenced.The sequencing of the RSus3 clones resulted in determination of 2700 bpupstream the ATG codon (see SEQ ID No. 1).

DNA Sequence Analysis

The sequence for the RSus3 promoter region was analysed for the presenceof putative cis-elements involved in the regulation of gene expression.The region upstream of the translational start codon was examined forthe conserved TATA and CAAT consensus sequences, and the region wasexamined for the presence of putative endosperm or seed specificelements.

Additionally the region between the TATA box and the translational startcodon was examined for the presence of intron donor and acceptor splicesites.

The translational start codon was chosen as basis for assigning bases inthe RSus3 promoter, i.e. the A in ATG was numbered +1 (E.g. thebase-pairs around the CAATGG are numbered −2, −1, +1, +2, +3 and +4).

Alignment of the RSus3 promoter region, with the promoter region regionfrom RSus1 (GenBank accession number: X59046) and from RSus2 (GenBankaccession number: X64770), gave no obvious similarity.

The results of the DNA sequence analysis are summarised in Table 1 (seeearlier). The GCN4 box and the three endosperm boxes are cis-elementsinvolved in endosperm specificity and the legumin boxes are cis-elementsinvolved in seed specificity. Additionally the −1072 bp endosperm boxand the GCN4 motif constitutes a putative prolamin element, which alsois involved in endosperm specificity, although the distance between theendosperm box and GCN4 motifs in RSus3 is greater than this (34 bp), thesequence shows high similarity to the consensus sequence.

The GCN4 box, the two endosperm boxes and the majority of the leguminboxes are located within 700 bp spanning from −1023 bp to −1707 bp. This700 bp sequence are located just upstream the TATA box.

A restriction map of the RSus3 promoter region is presented herein asFIG. 3.

Presentation of RSus3/GUS/NOSt Constructs

In this study, various RSus3 promoter sequences were fused with the uidAgene for generation of a series of RSus3/GUS expression cassettes.Specific deletions of the RSus3 promoter were constructed in an attemptto identify those parts of the promoter that are involved intissue-specificity. A series of tandem-repeat promoters were constructedin an attempt to enhance the promoter activity, and the intron 1 wasremoved from the promoter region, in order to establish whether it isnecessary for expression in guar endosperm.

The constructs were generated either by amplification of specificfragments by PCR or by subcloning. For verification of the resultingconstructs, control restriction digests, typically with HindIII, Nhe I,and NcoI were performed. Additionally, control PCR or sequencingreactions were performed in some cases.

Common features in all variants of the RSus3 promoter were,translational fusion to the uidA gene, presence of the TATA box, andconservation of region downstream of this (with the exception of thesingle construct from which the intron was removed). The series of RSus3promoter constructs centred around the upstream 700 bp region distal tothe TATA box. As described above, this sequence contains the majority ofputative cis-elements involved in specificity.

The various RSus3 promoter constructs are summarised in FIG. 13.

p2700 Construct (Plasmid Size 7938 bp)

An RSus3 promoter of 2700 bp was initially constructed. This constructconstituted the full-length RSus3 promoter, and was prepared byamplification of a 2700 bp XhoI/NcoI fragment from pTBR-ScaK3 intoSalI/NcoI in pGUSNOSt. Positive clones were verified by NcoI/HindIIIdigests, which resulted in 2 specific fragments, of 1160 bp and 1540 bp,in addition to the 5230 bp NcoI/HindIII fragment from the pGUSNOSt part.

p1730 Construct (Plasmid Size 6963 bp)

This 5′ truncation of RSus3 was constructed by generation of a 571 bpNheI/HindIII fragment spanning from −1160 bp to −1730 bp. After changingof the upstream HindIII site to a NheI site in p2700, the NheI/HindIIIfragment generated was subcloned into this p2700(upNheI). Positiveclones were identified by an NheI/HindIII control digest, and thisresulted in a specific fragment of same size as the subcloned fragment.

p1670 Construct (Plasmid Size 6907 bp)

This 5′ truncation was analogous to p1730, comprising a 515 bp insteadof 571 bp NheI/HindIII fragment, positive clones of which wereidentified in the same way.

p1560 Construct (Plasmid Size 6791 bp)

This 5′ truncations was also analogous to p1730, comprising a 400 bpinstead of 571 bp NheI/HindIII fragment, positive clones of which wereidentified in the same way.

p1450 Construct (Plasmid Size 6704 bp)

This construct was analogues to p2700, with ligation of a 1450 bpXhoI/NcoI fragment amplified from pSspK3 template, instead of the sameamplified from pScaK3. A NcoI/HindIII digest resulted in two specificbands of 1160 bp and 290 bp, besides the 5230 bp pGUSNOSt fragment.

p1160 Construct (Plasmid Size 6390 bp)

This truncated promoter constitutes removal of 1540 bp upstream theinternal HindIII site, and was generated by cutting p2700 with HindIII,followed by religation. Positive clones were identified by HindIIIdigest, which gave a single fragment in positive clones.

p2700-Prolamin Construct (Plasmid Size 7777 bp)

This internal truncation of 161 bp was produced by amplification of a1020 bp fragment comprising the TATA box to the ATG codon. This fragmentwas cloned in p2700(upNheI) as a HindIII/NcoI fragment. Positive cloneswere verified by a HindIII/NcoI digest, which resulted in a specificfragment of 1000 bp and a fragment of 6770 bp.

p1450-Intron (Plasmid Size 5837 bp)

In this construct, the intron 1 was removed by exactly splicing of theputative intron 1 acceptor and donor splice sites. This internaldeletion of the intron 1 was generated by amplification of a region ofp1450 which corresponded to RSus3 lacking the intron 1. After cloning inpCR2.1-TOPO, an XhoI/NcoI fragment containing this was subcloned in inpGUSNOSt. A positive clone was identified by restriction digestion withHindIII/NcoI, and the 3′ promoter end was verified by sequencing.

pTandem Legumin (Plasmid Size 7536 bp)

This tandem repeat promoter was constructed by generation of a 570 bpNheI fragment spanning from −1160 bp to −1730 bp by PCR. This fragmentwas subcloned in the NheI site in p1730. A positive clone was identifiedby control digests and a control PCR reaction with Nhedel1/Dellow1verified the orientation. The right orientation resulted in two bands of650 bp and 1300 bp, whereas the reverse orientation only resulted in a650 bp product.

pTandem Leg/Pro (Plasmid Size 7704 bp)

This tandem-repeat promoter was constructed by PCR amplifaction of a 740bp NheI fragment spanning form −990 bp to −1730 bp. This fragment wassubcloned in the NheI site in p1730. Positive clones were firstidentified by an Nhe I digest, and those of appropriate orientation weredetermined by HindIII digest. This was possible due to the internalHindIII site in the promoter, which was duplicated in this construct.Correct orientation resulted in a 740 bp HindIII fragment, whereas thereverse orientation resulted in a 1140 bp HindIII fragment.

pTandem Prolamin (Plasmid Size 7135 bp)

This tandem-repeat promoters was constructed by generation of a 170 bpHindIII fragment, spanning from −990 to −1160 bp by PCR, which wassubcloned in the HindIII site in p1730. A Positive clone were identifiedby control digests, and the right orientation was determined by controlPCR. Amplification with primer pair Nhedel1/HindGCN4 resulted in twobands of 600 bp and 750 bp for clones with right orientation, and tobands for of 300 bp and 750 bp for clones of reverse orientation.

See FIG. 15.

Transient Expression Experiments

The RSus3 expression cassettes described above were used to effect atransient transformation of guar tissue using the particle bombardmenttechnique.

These experiments served several purposes. Firstly, for testing theusefulness of the method for generation of transient transformed guartissue. Secondly, the purpose was to set up and optimise the ParticleInflow Gun (PIG) in Danisco Biotechnology, Holeby. Finally, the mainpurpose was to evaluate the strength and specificity of promoters, suchas RSus3, in guar tissues.

Control

By way of a control experiment, a control plasmid comprising the uidAgene fused to the ENOS promoter was constructed. It is known that theENOS promoter has high levels of expression in both endosperm and leaftissue.

Transient Expression of RSus3/GUS/NOSt in Guar Tissue

A total of 11 sets of particle bombardment experiments were performedfor testing the promoter strength and specificity of the RSus3 promoterregion. The above described RSus3/GUS/NOSt constructions had been testedin this period. The procedures for the bombardment, for the preparationof guar tissue and for the coating of gold particles are describedabove.

The first approach was to test whether the RSus3 promoter was active inguar endosperm or not. p2700 and p1450 were bombarded into guarendosperms, and the result showed significant expression of GUS inendosperm for both constructs, although the expression was lower thanfor the ENOS control. These results were so promising that thesubsequent work was concentrated around this promoter.

The second approach was to test whether the RSus3 was specific for theendosperm or whether it had expression in other parts of the plant. Theinitial experiment, which was performed on cotyledons from in vitrogrown seedlings, showed differences in expression between p2700 andp1450. p2700 had almost no expression of GUS in cotyledons, whereas somecotyledons transformed with p1450 displayed significantly higherexpression.

To test whether this expression pattern for p2700 and p1450 was the samein leaves, these constructs were also used to perform transienttransformation of juvenile leaves from mature plants. Thistransformation turned out to be difficult due to severe damage of theleaf tissue caused by the helium blast. Not only was the expression ofRSus3 concentrated in the vascular tissue of the leaves, but also theENOS control showed same expression pattern indicating that optimisationof the procedure for leaves were necessary. Due to limitations in time,however, these optimisation experiments were abolished after a while,and instead it was chosen exclusively to bombard cotyledons fromseedlings and from seeds.

The transient GUS expression, directed by the two RSus3 promoterconstructs, p2700 and p1450, justified further analysis of the promoterstrength and tissue specificity. As described above a series ofdeletions of the RSus3 promoter were constructed and these were testedin guar endosperm, guar embryo cotyledons and cotyledons from 12 and 20day old seedlings. Additionally, in an attempt to enhance the promoteractivity, 3 tandem promoters were constructed and the resultingconstructs were also tested in same tissue for evaluation of promoterstrength and specificity.

FIG. 16 presents transient GUS expression data from the control ENOSpromoter, the RSus3 promoter construct p1730 and the RSus3 promoterconstruct pTandem leg/pro (tandem repeat). The results, which are inaccordance with the findings for the other RSus3 promoter constructs,have been discussed above. These results show selective endospermexpression of an NOI.

The results of transient GUS expression in guar endosperms, afterbombardment with RSus3 constructs, are summarised in FIG. 17.

The data show that the promoter of the present invention causesselective expression of the NOI in endosperm. In addition, a tandemrepeat of the promoter of the present invention causes an increase inthe expression levels.

As can be seen at FIG. 17 the expression in guar endosperm are onlyslightly affected by deletion of the sequence between 2700 bp and 1160bp. The 1160 bp has still significant expression indicating that thecis-elements which caused the main part of expression in guar endospermare present between 1160 bp and the TATA box (÷989 bp). This part hasbeen removed in the p2700÷prolamin construct, and this result in a lossof 25% of the promoter activity compared to p2700.

pTandem leg/pro gives highest expression in endosperms, 3 times higherthan the 2700 construct and the level of expression is comparable to NOSexpression in endosperms.

No blue spots were observed when GUAR tissues were bombarded withmicroprojectiles treated in the same manner, only without DNA, and whenthe PIG was discharged without microprojectiles. Bombardment with thepromoterless GUS construct (pGUSNOSt) gave only a few blue spots,presumably caused by illegitimate recombination into the chromosomeadjacent to a promoter.

For comparative analysis of expression in endosperms versus cotyledons,values relative to the area of the bombarded tissue were calculated. Anaverage 4 weeks old endosperm comprises an area of 0.2 cm², and the samefor an average pre-emerged cotyledon. An average 12 days cotyledoncovers an area of 1.5 cm², and an average 20 days cotyledon an area of2.8 cm². Therefore the number of blue spots per 12 day or 20 dayscotyledon were corrected to number of blue spots per 0.2 cm².

FIG. 18 shows that the only highly significant change in promoterstrength was displayed by the Tandem leg/pro variant. Although thisconstruct directed a level of GUS expression which was three-fold largerthan that of the parent construct p2700, it retained specificity for theendosperm. Levels of expression in cotyledons was equally low with theTandem leg/pro variant as it was with p2700.

Co-Transformation

The largest component of variance in data obtained by measurements oftransient gene expression, following ballistic transformation, residesin the differences in the efficiency of delivery of the DNA to thetarget. It has been shown that one can compensate for this problem, andobtain more precise evaluation of transient gene expression, byco-transformation with a control expression vector containing adifferent reporter gene (Godon, C., Caboche, M, Daniel-Vedele, F. (1993)Biochimie 75: 591–595). By measuring the reporter activities andexpressing results as the ratio of the test activity to that of thecontrol, fluctuations that merely result from differences in the amountof DNA striking the target are accounted for.

The RSus3 promoter variants, were also tested in this manner. Thefirefly luciferase gene (luc) was fused to each of them, as a reporter,and ballistic transformation was repeated as described before, but twocontrol plasmids were included in equal molar amounts to the testplasmid. The strength and tissue-specificities of the different promoterconstructs were tested in modified transient expression studies ofballistically transfomed endosperms, and pre-emerged cotyledons, fromthe same guar seeds. Each experiment involved simultaneoustransformation with three separate plasmids. Of these, one was a testplasmid (p2700+intron/luc+, p2700−intron/luc+, pTandem leg/pro+intron/luc+, or pTandem leg/pro−intron/luc+ or pDB16/luc+), in whichpromoters were fused to the firefly luciferase gene (luc+) (FIGS. 19,20, 21, 22, and 23). The remaining two were control plasmids, in whicheither the uidA (GUS) gene or the renilla luciferase gene (pDB16/Rluc)were under the control of the NOS promoter (FIGS. 24 and 25). 2 μg oftest plasmid were mixed with 2 μg of each control plasmid, and coatingof particles with this DNA, preparation, bombardment, and subsequentincubation of tissue, were all performed as described above.

Enzyme extracts of bombarded tissues were made by grinding these inliquid nitrogen, and extracting the frozen macerate with a buffercontaining boric acid, that prevents gelling of the galactomannan incryopreserved material. Approximately 400 mg of each tissue wereextracted with 800 μl of extraction buffer (50 mM NaH₂PO₃, 50 mM H₂BO₃,1 mM dithiothreitol, 1 mM EDTA disodium salt, 10 mg/ml bovine serumalbumin, pH was adjusted to 7.0 by addition of sodium hydroxide).Samples were centrifuged at 12,000 g, at 4° C., for 2 minutes, and theresulting supernatants were used for analysis.

The activities of all three reporter genes were measuredluminometrically using a Turner TD-20/20 luminometer (Turner Designs,Sunnyvale, Calif., USA). The two luciferase acivities were measuredsequentially in the same reaction mixture, after the ‘Dual-Luciferase®’method described in Promega Technical Manual No. 40 (PromegaCorporation, Madison, Wis., USA). 20 μl of guar tissue extract wererapidly mixed with 100 μl of LARII reagent in the apparatus, which after2 seconds measured and integrated the light evolved over the subsequent10 seconds. Immediately after this, 100 μl of ‘Stop and Glo®’ reagentwere added to the reaction mixture and the renilla luciferaseluminescence was measured in the same manner.

GUS activity was measured according to the GUS-Light™ system instructionmanual (TROPIX Inc., Bedford, Mass., USA). 20 μl of guar tissue extractwere added to 180 μl of GUS reaction buffer, and incubated at 25° C. forone hour. The tube was placed in the luminometer, 300 μl of ‘lightemission accelerator’ were added, and after 2 seconds the light emittedduring the subsequent 10 seconds was measured and integrated.

The values obtained are summarised in the table below.

Relative Integrated Light Signal Dual Luciferase ® Reporter AssayGUS-Light ™ system (Promega) (Tropix) Endosperm Cotyledon EndospermCotyledon Rsus3/luciferase experiment luc+ Rluc luc+ Rluc GUS GUSp2700 + intron/luc+ 7.7 352.0 1.2 248.7 194.5 208.2 (pDB16/Rluc andpDB16/GUS) 7.6 350.4 0.8 236.4 165.3 192.4 7.6 353.2 0.8 256.7 189.8206.7 P2700 − intron/luc+ 20.9 574.0 0.6 185.2 269.7 215.0 (pDB16/Rlucand pDB16/GUS) 20.9 585.1 0.5 179.3 252.1 151.3 21.2 596.7 0.5 195.1228.0 PTandem (leg/pro) + intron/luc+ 13.9 249.5 1.1 209.2 93.4 166.4(pDB16/Rluc and pDB16/GUS) 13.3 248.8 0.9 202.3 99.6 142.0 13.3 247.50.8 206.6 104.9 182.7 pTandem (leg/pro) − intron/luc+ 42.8 570.1 1.7205.1 249.2 172.3 (pDB16/Rluc and pDB16/GUS) 43.2 591.8 1.5 202.6 225.2156.3 44.7 604.3 1.5 213.9 167.2 pDB16/luc+ 45.3 565.5 22.3 155.5 289.8206.6 (pDB16/Rluc and pDB16/GUS) 43.4 582.0 21.4 176.2 205.8 191.8 45.1623.4 19.9 172.4 333.2 206.4

The same results are expressed in FIG. 26 as ‘relative strength ofpromoter’, the value of which is calculated by the division of tworatios, the test ratio and the control ratio. The test ratio wascalculated by dividing the luminescence generated by the fireflyluciferase, under control of test promoter construct, by the valueobtained from the control reporter gene (either uidA or Rluc) under thecontrol of the NOS promoter. The control ratio was calculated bydividing the luminescence from the firefly luciferase by the value fromthe control reporter gene (either uidA or Rluc) when both genes wereunder the control of the NOS promoter. ‘Relative strength of promoter’was calculated by dividing the test ratio by the control ratio andmultiplying the value obtained by one hundred.

The results in FIG. 26 show a pattern that mirrors that shown in FIG. 17insofar as the Tandem leg/pro configuration enhances the activity of thepromoter while retaining tissue specificity. Although it appeared fromthe earlier results that the Tandem leg/pro promoter construct was twiceas strong as the NOS promoter strength in endosperm tissue, the resultsin FIG. 26 show that these two promoters in fact display similarstrength in this tissue. These results also show that removal of theintron from constructs seems to enhance promoter strength in guartissue. This is a preferred aspect for some embodiments of the presentinvention. Hence, preferably the NOI does not comprise an intron.

SUMMARY

In summation, the present invention relates to a promoter and, also to aconstruct comprising the same. In particular the present inventionrelates to the use of a promoter for the expression of a NOI in anorganism.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

REFERENCES

-   Bloomfield V A (1991) Condensation of DNA by multivalent Cations:    Considerations on Mechanism Biopolymers 31: 1471–1481-   Donovan C R & Lee J W (1977) The Growth of Detached Wheat Heads in    Liquid Culture Plant Science Letters 9: 107–113-   Kikkert J R (1993) The Biolistic PDS-1000/He device Plant cell,    Tissue and Organ Culture 33: 221–226-   Knudsen S & Müller M (1991) Transformation of the developing barley    endosperm by particle bombardment Planta 185: 330–336-   Siebert PD, Chenchik A, Kellogg D E, Lukyanov K A and Lukyanov    SA (1995) An improved PCR method for walking in uncloned genomic DNA    Nucleic Acids Research, vol. 23, no.6; p. 1087–1088-   Huang JW, Chen J T, Yu W P, Shyur L F, Wang A Y, Sung H Y, Lee P D    and Su (1996) Complete Structures of Three Rice Sucrose Synthase    Isogenes and Differential Regulation of Their Expressions Biosci.    Biotech. Biochem. 60(2): p233–239-   Copeland L (1990) Methods in plant biochemistry Vol. 3, Kap. 4:    Enzymes of Sucrose Metabolism Academic Press Limited-   Dörmann P & Benning C (1998) The role of UDP-glucose epimerase in    carbohydrate metabolism of Arabidobsis The Plant Journal 13(5):    641–652-   Finer J J, Vain P, Jones M W & McMullen M D (1992) Development of    the particle inflow gun for DNA delivery to plant cells Plant cell    Reports 11: 323–328-   Jefferson R A (1987) Assaying Chimeric Genes in Plant: The GUS Gene    Fusion System Plant Molecular Biology Reporter vol. 5, number 4-   Karrer E E & Rodriquez R L (1992) Metabolic regulation of rice    alpha-amylase and sucrose synthase genes in planta Plant J 2(4):    517–523-   Klein T M, Wolf E D, Wu R & Sanford J C (1987) High-velocity    microprojectiles for delivery nucleic acids into living cells Nature    327: 70–73-   Klein T M, Fromm M, Weissinger A, Tomes D, Schaaf S, Sletten M &    Sanford J C (1988) Transfer of foreign genes into intact maize cells    with high-velocity microprojectiles Proc. Natl. Acad. Sci. USA 85:    4305–4309-   Lopes M A & Larkin B A (1993) Endosperm origin, development, and    function The Plant Cell 5: 1383–1399-   Sanford J C, Klein T M, Wolf E D & Allen N (1987) Delivery of    substances into cells and tissues using a particle bombardment    process Particulate Science and Technology 5: 27–37-   Thomas T L (1993) Gene expression during plant embryogenesis and    germination: An overview The Plant Cell 5: 1401–1410-   Vain P, Keen N, Murillo J, Rathus C, Nemes C & Finer J J (1993)    Development of the Particle Inflow Gun Plant cell, Tissue and Organ    Culture 33: 237–246-   West M A L & Harada J J (1993) Embryogenesis in higher plants: An    overview The Plant Cell 5: 1361–1369-   Whistler R L & Hymowitz T (1979) GUAR:Agronomy, Production,    Industrial use and Nutrition Purdue University Press, West    Lafayette, Ind.

REFERENCES FOR TABLE 1

-   Bäumlein H, Nagy I, Villarroel R, Inzé D & Wobus U (1992)    Cis-analysis of a seed protein gene promoter: the conservative    RY-repeat CATGCATG within the legumin box is essential for    tissue-specific expression of a legumin gene The Plant Journal 2(2):    233–239-   Joshi C P (1987) An inspection of the domain between putative TATA    box and translation start site 79 plant genes Nuc. Acid. Res.    15(16): 6643–6653-   Marzabal P, Busk P K, Ludevid M D & Torrent M (1998) The bifactorial    endosperm box of gamma-zein gene: characterisation and function of    the Pb3 and GZM cis-acting elements. The Plant Journal 16(1): 41–52-   Müller M & Knudsen S (1993) The nitrogen response of a barley    C-hordein promoter is controlled by positive and negative regulation    of the GCN4 and endosperm box The Plant Journal 4(2): 343–355-   Simpson C G & Filipowicz W (1996) Splicing of precursors to mRNA in    higher plants: mechanism, regulation and sub-nuclear organisation of    the spliceosomal machinery Plant Molecular Biology 32: 1–41

1. An isolated promoter comprising a nucleotide sequence correspondingto that shown as SEQ ID No.
 6. 2. An isolated promoter having anucleotide sequence corresponding to that shown as SEQ ID No.
 6. 3. Anisolated promoter comprising a nucleotide sequence corresponding to thatshown in SEQ ID No.
 1. 4. An isolated promoter having a nucleotidesequence corresponding to that shown in SEQ ID No.
 1. 5. A promoteraccording to claim 1, wherein the promoter is obtained from a plant ofthe genus Oryza.
 6. A promoter according to claim 1, wherein thepromoter is operably linked to a nucleotide sequence of interest.
 7. Apromoter according to claim 3, wherein the promoter is linked to thesequence presented as SEQ ID No. 2, or a nucleotide sequence with atleast 95% homology to SEQ ID No.
 2. 8. A promoter according to claim 7,wherein if a nucleotide sequence of interest is operably linked to thepromoter then the sequence presented as SEQ ID No. 2, or a nucleotidesequence with at least 95% homology to SEQ ID No. 2, is locatedintermediate the promoter and the nucleotide sequence of interest.
 9. Apromoter according to claim 1, wherein the promoter is linked to thesequence presented as SEQ ID No.
 5. 10. A promoter according to claim 9,wherein if a nucleotide sequence of interest is operably linked to thepromoter then the sequence presented as SEQ ID No. 5 is locatedintermediate the promoter and the nucleotide sequence of interest.
 11. Aconstruct comprising the promoter according to claim 1, wherein thepromoter is operably linked to a nucleotide sequence of interest.
 12. Anexpression vector comprising the promoter according to claim
 1. 13. Atransformation vector comprising the promoter according to claim
 1. 14.A transformed host or host cell comprising the promoter according toclaim
 1. 15. A transformed host or host cell according to claim 14,wherein the host or host cell is a plant or a plant cell.
 16. A methodof preparing a protein of interest, the method comprising expressing anucleotide sequence of interest which encodes at least a part of theprotein of interest, wherein the nucleotide sequence of interest isoperably linked to the promoter according to claim 1, optionallyisolating the expression product of the nucleotide sequence of interest,forming the protein of interest if the expression product of thenucleotide sequence of interest is not all of the protein of interest,optionally isolating the protein of interest.
 17. A method according toclaim 16 wherein the nucleotide sequence of interest codes for all ofthe protein of interest.
 18. A method for expressing a nucleotidesequence of interest in endosperm, the method comprising expressing inendosperm the nucleotide sequence of interest operably linked to thepromoter according to claim 1, wherein the endosperm is transgenic forthe nucleotide sequence of interest operably linked to the promoteraccording to claim
 1. 19. An isolated promoter sequence obtained fromDeposit No. NCIMB
 41011. 20. The promoter of claim 3, wherein thepromoter is linked to the sequence presented as SEQ ID No. 2, or anucleotide sequence with at least 98% homology to SEQ ID No.
 2. 21. Thepromoter of claim 1, wherein the promoter is linked to the sequencepresented as SEQ ID No.
 4. 22. The promoter of claim 1, wherein thepromoter is linked to the sequence presented as SEQ ID No. 2, or anucleotide sequence with at least 95% homology to SEQ ID No.
 2. 23. Thepromoter of claim 1, wherein the promoter is linked to the sequencepresented as SEQ ID No. 2, or a nucleotide sequence with at least 98%homology to SEQ ID No.
 2. 24. The promoter of claim 21, wherein thepromoter is linked to the sequence presented as SEQ ID No. 2, or anucleotide sequence with at least 95% homology to SEQ ID No.
 2. 25. Thepromoter of claim 21, wherein the promoter is linked to the sequencepresented as SEQ ID No. 2, or a nucleotide sequence with at least 98%homology to SEQ ID No.
 2. 26. The promoter of claim 1, wherein thepromoter is linked to the sequence presented as SEQ ID No.
 5. 27. Thepromoter of claim 21, wherein the promoter is linked to the sequencepresented as SEQ ID No.
 5. 28. The promoter of claim 22, wherein thepromoter is linked to the sequence presented as SEQ ID No.
 5. 29. Thepromoter of claim 23, wherein the promoter is linked to the sequencepresented as SEQ ID No.
 5. 30. The promoter of claim 24, wherein thepromoter is linked to the sequence presented as SEQ ID No.
 5. 31. Thepromoter of claim 25, wherein the promoter is linked to the sequencepresented as SEQ ID No. 5.